Gambogic acid, analogs and derivatives as activators of caspases and inducers of apoptosis

ABSTRACT

The present invention is directed to gambogic acid, analogs and derivatives thereof, represented by the general Formulae I-III:                    
     wherein R 1 -R 5  are defined herein. The present invention also relates to the discovery that compounds having Formula I-III are activators of caspases and inducers of apoptosis. Therefore, the activators of caspases and inducers of apoptosis of this invention can be used to induce cell death in a variety of clinical conditions in which uncontrolled cell growth and spread of abnormal cells occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/135,424, filed May 21, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to gambogic acid, novel analogs of gambogic acidand derivatives of gambogic acid, and the discovery that these compoundsare activators of caspases and inducers of apoptosis. The invention alsorelates to the use of these compounds as therapeutically effectiveanti-cancer agents.

2. Description of Background Art

Organisms eliminate unwanted cells by a process variously known asregulated cell death, programmed cell death or apoptosis. Such celldeath occurs as a normal aspect of animal development as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis, et al., Dev. 112:591-603 (1991); Vaux, et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published applicationWO96/20721).

There are a number of morphological changes shared by cells experiencingregulated cell death, including plasma and nuclear membrane blebbing,cell shrinkage (condensation of nucleoplasm and cytoplasm), organellerelocalization and compaction, chromatin condensation and production ofapoptotic bodies (membrane enclosed particles containing intracellularmaterial) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).

Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wyllie, et al., Int. Rev. Cyt.68:251 (1980); Ellis, et al., Ann. Rev. Cell Bio. 7:663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

It has been found that a group of proteases are a key element inapoptosis (see, e.g. Thomberry, Chemistry and Biology 5:R97-R103 (1998);Thomberry, British Med. Bull. 53:478-490 (1996)). Genetic studies in thenematode Caenorhabditis elegans revealed that apoptotic cell deathinvolves at least 14 genes, two of which are the pro-apoptotic(death-promoting) ced (for cell death abnormal) genes, ced-3 and ced-4.CED-3 is homologous to interleukin 1 beta-converting enzyme, a cysteineprotease, which is now called caspase-1. When these data were ultimatelyapplied to mammals, and upon further extensive investigation, it wasfound that the mammalian apoptosis system appears to involve a cascadeof caspases, or a system that behaves like a cascade of caspases. Atpresent, the caspase family of cysteine proteases comprises 14 differentmembers, and more may be discovered in the future. All known caspasesare synthesized as zymogens that require cleavage at an aspartyl residueprior to forming the active enzyme. Thus, caspases are capable ofactivating other caspases, in the manner of an amplifying cascade.

Apoptosis and caspases are thought to be crucial in the development ofcancer (Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds.,Humana Press (1999)). There is mounting evidence that cancer cells,while containing caspases, lack parts of the molecular machinery thatactivates the caspase cascade. This makes the cancer cells lose theircapacity to undergo cellular suicide and the cells become cancerous. Inthe case of the apoptosis process, control points are known to existthat represent points for intervention leading to activation. Thesecontrol points include the CED-9—BCL-like and CED-3—ICE-like gene familyproducts. These are intrinsic proteins that regulate the decision of acell to survive or die and they execute part of the cell death processitself (see Schmitt, et al., Biochem. Cell. Biol. 75:301-314 (1997)).BCL-like proteins include BCL-xL and BAX-alpha, which appear to functionupstream of caspase activation. BCL-xL appears to prevent activation ofthe apoptotic protease cascade, whereas BAX-alpha accelerates activationof the apoptotic protease cascade.

It has been shown that chemotherapeutic (anti-cancer) drugs can triggercancer cells to undergo suicide by activating the dormant caspasecascade. This may be a crucial aspect of the mode of action of most, ifnot all, known anticancer drugs (Los et al., Blood, Vol. 90, No8:3118-3129 (1997); Friesen, et al., Nat. Med. 2:574 (1996)). Themechanism of action of current antineoplastic drugs frequently involvesan attack at specific phases of the cell cycle. In brief, the cell cyclerefers to the stages through which cells normally progress during theirlifetimes. Normally, cells exist in a resting phase termed G_(o). Duringmultiplication, cells progress to a stage in which DNA synthesis occurs,termed S. Later, cell division, or mitosis occurs, in a phase called M.Antineoplastic drugs such as cytosine arabinoside, hydroxyurea,6-mercaptopurine, and methotrexate are S phase specific, whereasantineoplastic drugs such as vincristine, vinblastine, and paclitaxelare M phase specific. Many antineoplastic drugs slow growing tumors. Forexample, colon cancers exist -primarily in the G_(o) phase, whereasrapidly proliferating normal tissues, for example bone marrow, existprimarily in the S or M phase. Thus, a drug like 6-mercaptopurine cancause bone marrow toxicity while remaining ineffective toward a slowgrowing tumor. Other aspects of the chemotherapy of neoplastic diseasesare known to those skilled in the art (see, e.g., Hardman, et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, NinthEdition, McGraw-Hill, New York (1996), pp. 1225-1287). Thus, it is clearthat the possibility exists for the activation of the caspase cascade,although the exact mechanisms for doing so are not clear at this point.It is equally clear that insufficient activity of the caspase cascadeand consequent apoptotic events are implicated in various types ofcancer. The development of caspase cascade activators and inducers ofapoptosis is a highly desirable goal in the development oftherapeutically effective antineoplastic agents. Moreover, sinceautoimmune diseases and certain degenerative diseases also involve theproliferation of abnormal cells, therapeutic treatment for thesediseases could also involve the enhancement of the apoptotic processthrough the administration of appropriate caspase cascade activators andinducers of apoptosis.

Gambogic acid was isolated from gamboge and the structure was deducedfrom the ¹H NMR spectrum and by comparison with morellin, which also hasthe xanthone core of gambogic acid (Ahmad, S. A., et al. J. Chem. Soc.(C) 772-779 (1966); Ollis, W. D., et al. Tetrahedron, 21:1453-1470(1965).

Asano J., et al., Phytochemistry, 41:815-820 (1996), reported theisolation of several xanthones, including gambogic acid from gamboge.They reported that gambogic acid is cytotoxic to both HeLa and HELcells.

Lin, L. -J., et al., Magn. Reson. Chem. 31:340-347 (1993), reported theisolation of gambogic acid, as well as isogambogic acid andisomorellinol. All three compounds were reported to be cytotoxic againstKB and KB-V1 cell lines.

SUMMARY OF THE INVENTION

The present invention is related to the discovery that gambogic acid,its analogs and derivatives, as represented in Formulae I-III, areactivators of the caspase cascade and inducers of apoptosis. Thereforethe first aspect of the present invention is directed to the use ofcompounds of Formulae I-III as inducers of apoptosis.

A second aspect of the present invention is to provide a method fortreating, preventing or ameliorating neoplasia and cancer byadministering a compound of Formulae I-III to a mammal in need of suchtreatment.

A number of compounds within the scope of the present invention arenovel compounds. Therefore, a third aspect of the present invention isto provide novel compounds of Formulae I-III, and to also provide forthe use of these novel compounds for treating, preventing orameliorating neoplasia and cancer.

A fourth aspect of the present invention is to provide a pharmaceuticalcomposition useful for treating disorders responsive to the induction ofapoptosis, containing an effective amount of a compound of FormulaeI-III in admixture with one or more pharmaceutically acceptable carriersor diluents.

A fifth aspect of the present invention is directed to methods for theisolation and preparation of novel compounds of Formulae 1-111.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict photographs of T47D human breast cancer cells treatedwith gambogic acid: control cells (FIG. 1A); cells treated with 2.5 μMof gambogic acid for 2 h (FIG. 1B); cells treated with 2.5 μM ofgambogic acid for 6 h (FIG. 1C).

FIGS. 2A-B depict fluorescent photographs of T47D human breast cancercells treated with gambogic acid and stained with a fluorescent DNAprobe: control cells (FIG. 2A); cells treated with 10 μM of gambogicacid for 24 h (FIG. 2B).

FIGS. 3A-C depict photographs of Jurkat leukemia cells treated withgambogic acid: control cells (FIG. 3A); cells treated with 10 μM ofgambogic acid for 30 min (FIG. 3B); cells treated with 10 μM of gambogicacid in the presence of 10 μM of caspase inhibitor cbz-Val-Asp-fink(FIG. 3C).

FIG. 4 depicts the caspase activity in T47D human breast cancer cellsand MRC5 human non-transformed fibroblast cells treated for 2 h withdifferent concentrations of gambogic acid.

FIGS. 5A-D depict western blots of poly(ADP)ribose polymerase (PARP)cleavage. FIG. 5A, Jurkat leukemia cells: (a) DMSO control, (b) treatedwith 1 μM of staurosporine for 2 h, (c) inactive control, (d) treatedwith 2.5 μM of gambogic acid for 2 h. FIG. 5B, HL-60 human leukemiacancer cells: (a) DMSO control, (b) treated with 1 μM of staurosporinefor 2 h, (c) inactive control, (d) treated with 2.5 μM of gambogic acidfor 2 h. FIG. 5C, T47D human breast cancer cells: (a) DMSO control, (b)treated with 1 μM of staurosporine for 2 h, (c) treated with 2.5 μM ofgambogic acid for 2 h, (d) treated with 5 μM of gambogic acid for 2 h,(e) DMSO control, (f) treated with 1 μM of staurosporine for 4 h, (g)treated with 2.5 μM of gambogic acid for 4 h, (h) treated with 5 μM ofgambogic acid for 4 h. FIG. 5D, PC3 human prostate cancer cells: (a)DMSO control, (b) treated with 1 μM of staurosporine for 2 h, (c)treated with 2.5 μM of gambogic acid for 2 h, (d) treated with 5 μM ofgambogic acid for 2 h, (e) DMSO control, (f) treated with 1 μM ofstaurosporine for 4 h, (g) treated with 2.5 μM of gambogic acid for 4 h,(h) treated with 5 μM of gambogic acid for 4 h.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises out of the discovery that gambogic acid isa potent and highly efficaceous activator of the caspase cascade andinducer of apoptosis. Therefore gambogic acid is useful for treatingdisorders responsive to induction of apoptosis.

There are many functional groups in the structure of gambogic acid whichcan be modified. These include, but are not limited to, the carboxylgroup, which can be converted to an ester, amide, ketone or alcohol andother functional groups; the ester and amide, in turn, may also containother functional groups, such as the carboxyl of an amino acid, whichcan be further modified; the hydroxy group, may be converted to anether, ester or other functional groups; the carbon-carbon double bondbetween C-9 and C-10 is part of an α,β-unsaturated ketone, which canreact with a nucleophile, be reduced to a carbon-carbon single bond, ormay be converted to an epoxide, which in turn may undergo furtherreaction; the carbon-carbon double bond between C-27 and C-28 is part ofan α,β-unsaturated carboxyl, that may also react with a nucleophile, bereduced to a carbon-carbon single bond, or may be converted to acyclopropane ring, which in turn may undergo further reaction; the twoisoprene carbon-carbon double bonds at C-37/C-38 and C-32/C-33, may alsobe reduced to a carbon-carbon single bond, be cleaved to form analdehyde group or a carboxyl group, both of which may be modified toother functional groups, or be converted to an epoxide, which in turnmay undergo further reaction; the carbon-carbon double bond between C-3and C-4 may also be reduced to a carbon-carbon single bond, or beconverted to an epoxide that may undergo further reaction; the ketonegroup at C-12 may be reduced to an alcohol, or may be converted to anoxime, a semicarbazone, or an amino group; the other ketone group mayalso be reduced, or may be converted to other functional groups. Inshort, many derivatives of gambogic acid can be prepared.

In addition, analogs of gambogic acid, including isomorellin, morellicacid, desoxymorellin, gambogin, morelline dimethyl acetal, isomoreollinB Moreollic acid, gambogenic acid, gambogenin, isogambogenin,desoxygambogenin, gambogenin dimethyl acetal, gambogellic acid, hanburin(Asano, J., et al., Phytochemistry 41:815-820 (1996)), isogambogic acid,isomorellinol (Lin, L. -J., et al., Magn. Reson. Chem. 31:340-347(1993)) and neo-gambogic acid (Lu, G. B., et al., Yao Hsueh Hsueh Pao19:636-639 (1984)) can be isolated from gamboge. Other analogs ofgambogic acid, including morellin, desoxymorellin, dihydroisomorellin(Bhat et al. Indian J. Chem. 2:405-409 (1964)) and moreollin (Rao et al.Proc. Indian Acad. Sci. 87A:75-86 (1978)), can be isolated from the seedof Garcinia morella. Morellinol can be isolated from the bark ofGarcinia morella (Adawadkar et al. Indian J. Chem. 14B:19-21 (1976)).Gaudichaudiones (A-H) and gaudichaudiic acids A-E can be isolated fromthe leaves of Garcinia gaudichaudii (Guttiferae) (Cao, S. -G., et al.,Tetrahedron 54(36):10915-10924 (1998) and Cao, S. -G., et al.,Tetrahedron Lett. 39(20):3353-3356 (1998)), and forbesione can beisolated from Garcinia forbesii (Leong, Y. -W., et al., J. Chem. Res.,Synop. 392-393 (1996)).

The present invention, therefore, also arises out of the discovery thatnovel derivatives and analogs of gambogic acid are also activators ofthe caspase cascade and inducers of apoptosis. Therefore thesederivatives and analogs of gambogic acid are useful for treatingdisorders responsive to the induction of apoptosis.

Specifically, compounds useful in this aspect of the present inventionare gambogic acid, its analogs and derivatives as represented byFormulae I-III:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

the dotted lines are single bonds, double bonds or an epoxy group;

X together with the attached carbon is a methylene, carbonyl,hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, a hydrazone,an arylhydrazone or semicarbazone;

Y together with the attached carbon is a methylene, carbonyl,hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, a hydrazone,an arylhydrazone or semicarbazone;

R₁ is formyl, methylenehydroxy, carboxy, acyl (R_(a)CO), optionallysubstituted alkoxycarbonyl (R_(a)OCO), optionally substitutedalkylthiocarbonyl, optionally substituted aminocarbonyl (carbamyl,R_(b)R_(c)NCO) or hydroxyaminocarbonyl, where R_(a) is hydrogen,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted lower aralkyl group or N-succinimidyl; R_(b) andR_(c) are independently hydrogen, optionally substituted heteroalkyl,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group, including piperidine,morpholine and piperazine.

R₂ is hydrogen, optionally substituted alkyl, acyl (R_(a)CO), carbamyl(R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) and R_(c) aredefined above; R_(d) is hydrogen, optionally substituted lower alkyl,optionally substituted aryl, or optionally substituted lower aralkylgroups;

R₃ is hydrogen or prenyl;

R₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino;

R₅ is hydrogen, optionally substituted alkyl or acyl (R_(a)CO), carbamyl(R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b), R_(c) andR_(d) are defined above.

Preferred compounds falling within the scope of Formula I includecompounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxycarbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamoyl,1-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)ethylcarbamyl or N-morpholinylcarbonyl; R₂ is hydrogen,formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinyl-carbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; Xand Y is O; R₃ is prenyl; and the dotted lines are double bonds or anepoxy group. If the double bond is present at C27-28, it is preferredthat it has the Z configuration.

Preferred compounds falling within the scope of Formula II includecompounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxycarbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)ethylcarboxy or N-morpholinylcarbonyl; R₂ is hydrogen,formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl;and R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy, hydrogen,methoxy, ethoxy, methylthio, ethylthio, butylthio, dimethylarnino,diethylamino, piperidinyl, pyrrolidinyl, imidazolyl, pyrazolyl,N-methylpiperazinyl, 2-(dimethylamino)ethylamino or morpholinyl; X and Yis O; R₃ is prenyl; and the dotted lines are double bonds. If the doublebond is present at C27-28, it is preferred that it has the Zconfiguration.

Preferred compounds falling within the scope of Formula III includecompounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxycarbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)ethylcarbamyl or N-morpholinylcarbonyl; R₂ is hydrogen,formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; R₅is hydrogen, formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; Xand Y is O; R₃ is prenyl; and the dotted lines are double bonds. If thedouble bond is present at C27-28, it is preferred that it has the Zconfiguration.

Exemplary preferred compounds that may be employed in the method ofinvention include, without limitation:

Gambogic acid;

Methyl gambogate;

9,10-Dihydrogambogic acid;

9,10-Dihydrogambogyl (4-methylpiperazine);

9,10-Dihydrogambogyl (2-dimethylaminoethylamine);

Gambogyl diethylamine;

Gambogyl dimethylamine;

Gambogyl amine;

Gambogyl hydroxyamine;

Gambogyl piperidine;

6-Methoxy-gambogic acid;

6-(2-Dimethylaminoethoxy)-gambogic acid;

6-(2-Piperidinylethoxy)-gambogic acid;

6-(2-Morpholinylethoxy)-gambogic acid;

6-Methoxy-gambogyl piperidine;

Gambogyl morpholine;

Gambogyl (2-dimethylaminoethylamine);

10-Morpholinyl-gambogyl morpholine;

10-Morpholinyl-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl morpholine;

10-Piperidinyl-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl (4-methylpiperazine);

Gambogyl (4-methylpiperazine);

Methyl-6-methoxy-gambogate;

Gambogenic acid;

Gambogenin;

10-Methoxy-gambogic acid;

10-Butylthio-gambogic acid;

10-(4-Methylpiperazinyl)-gambogic acid;

10-Pyrrolidinyl-gambogic acid;

Methyl-10-Morpholinyl-gambogate;

10-Piperidinyl-gambogic acid;

10-Morpholinyl-gambogic acid;

N-(2-Gambogylamidoethyl)biotinamide;

Gambogyl (2-morpholinylethylamine);

9,10-Epoxygambogic acid;

Gambogyl (4-(2-pyridyl)piperazine);

10-(4-(2-Pyridyl)piperazinyl)gambogyl (4-(2-pyridyl)piperazine);

6-Acetylgambogic acid;

10-(4-(2-Pyridyl)piperazinyl)gambogic acid;

N-Hydroxysuccinimidyl gambogate;

8-(Gambogylamido)octanoic acid;

6-(Gambogylamido)hexanoic acid;

12-(Gambogylamido)dodecanoic acid;

N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;

N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;

N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate;

10-Methoxy-gambogyl piperidine;

Gambogyl (4-(2-pyrimidyl)piperazine);

Gambogyl (bis(2-pyridylmethyl)amine);

Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine);

Gambogyl (4-benzylpiperazine);

Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);

Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine);

Gambogyl (N-methyl-8-(methylamino)-3,6-dioxaoctylamine);

Gambogyl (N-ethyl-2-(ethylamino)ethylamine);

Gambogyl (4-isopropylpiperazine);

Gambogyl (4-cyclopentylpiperazine);

Gambogyl (N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine);

Gambogyl (2,5-dimethylpiperazine);

Gambogyl (3,5-dimethylpiperazine);

Gambogyl (4-(4-acetylphenyl)piperazine);

Gambogyl (4-ethoxycarbonylpiperazine);

Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine);

Gambogyl (4-(2-hydroxyethyl)piperazine);

Gambogyl (N-methyl-2-(methylamino)ethylarnine);

Gambogyl (N-methyl-2-(benzylamino)ethylamine);

Gambogyl (N-methyl-(6-methyl-2-pyridyl)methylamine);

Gambogyl (N-ethyl-2-(2-pyridyl)ethylamine);

Gambogyl (N-methyl-(2-pyridyl)methylamine);

Gambogyl (N-methyl-4-(3-pyridyl)butylamine);

Gambogyl (bis(3-pyridylmethyl)amine);

Gambogyl (2,4-dimethyl-2-imidazoline);

Gambogyl (4-methyl-homopiperazine);

Gambogyl (4-(5-hydroxy-3-oxapentyl)piperazine);

Gambogyl (3-dimethylaminopyrrolidine);

Gambogyl ((2-furanyl)methylamine);

Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine);

Gambogyl (3,4,5-trimethoxybenzylamine);

Gambogyl (2-(2-methoxyphenyl)ethylamine);

Gambogyl (2-methoxybenzylamine);

Gambogyl (3,4-methylenedioxybenzylamine);

Gambogyl (2-(2,5-dimethoxyphenyl)ethylamine);

Gambogyl (2-(3-methoxyphenyl)ethylamine);

Gambogyl (3-(piperidinyl)propylamine);

Gambogyl (2-(piperidinyl)ethylamine);

Gambogyl (3,4-dimethoxybenzylamine);

Gambogyl ((2-tetrahydrofuranyl)methylamine);

Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine);

Gambogyl (2-diethylaminoethylamine);

Gambogyl (2,2-dimethyl-3-dimethylaminopropylamine);

Gambogyl ((N-ethoxycarbonyl-4-piperidinyl)amine);

Gambogyl (2-carbamylpyrrolidine);

Gambogyl (3-(homopiperidinyl)propylamine);

Gambogyl ((N-benzyl-4-piperidinyl)amine);

Gambogyl (2-(4-methoxyphenyl)ethylamine);

Gambogyl (4-oxa-hex-5-enylamine);

Ganbogyl (6-hydroxyhexylamine);

Gambogyl (2-(3,5-dimethoxyphenyl)ethylamine);

Gambogyl (3,5-dimethoxybenzylamine); and

Gambogyl (2-carbamyl-2-(4-hydroxyphenyl)ethylamine).

The positions in gambogic acid are numbered according to Asano, J., etal., Phytochemistry 41:815-820 (1996), and Lin, L. -J., et al., Magn.Reson. Chem. 31:340-347 (1993).

The present invention is also directed to novel compounds within thescope of Formulae I-III. These compounds include compounds of Formula Iwherein if R₁ is carboxy or methoxycarbonyl and X and Y are O, then R₂is not hydrogen or methyl. These compounds also include compounds ofFormula II wherein if R₁ is formyl or carboxy, R₂ is hydrogen, R₃ ishydrogen and X and Y are O, then R₄ is not methoxy or ethoxy. Thesecompounds also include compounds of Formula III wherein if R₁ is formylor carboxy and X and Y are O, then at least one of R₂ or R₅ are nothydrogen.

Exemplary preferred compounds that may be employed in this inventioninclude, without limitation:

9,10-Dihydrogambogyl (4-methylpiperazine);

9,10-Dihydrogambogyl (2-(dimethylamino)ethylamine);

9,10-Dihydro-1 2-hydroxygambogic acid;

Gambogyl diethylamine;

Gambogyl dimethylamine;

Gambogyl amine;

Gambogyl hydroxyamine;

Gambogyl piperidine;

6-Methoxy-gambogic acid;

6-(2-Dimethylaminoethoxy)-gambogic acid;

6-(2-Piperidinylethoxy)-gambogic acid;

6-(2-Morpholinylethoxy)-gambogic acid;

6-Methoxy-gambogyl piperidine;

Gambogyl 4-morpholine;

Gambogyl 2-(dimethylamino)ethylamine;

10-Morpholinyl-gambogyl morpholine;

10-Morpholinyl-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl morpholine;

10-Piperidinyl-gambogyl piperidine;

10-(4-Methylpiperazinyl)-gambogyl (4-methylpiperazine);

Gambogyl (4-methylpiperazine);

10-Methoxy-gambogic acid;

10-Butylthio-gambogic acid;

10-(4-Methylpiperazinyl)-gambogic acid;

10-Pyrrolidinyl-gambogic acid;

Methyl-10-Morpholinyl-gambogate;

10-Piperidinyl-gambogic acid;

10-Morpholinyl-gambogic acid;

10-Cyclohexyl-gambogic acid;

10-Methyl-gambogic acid;

N-(2-Gambogylamido-ethyl)biotinamide;

Gambogyl (2-(4-morpholinyl)ethylamine);

9,10-Epoxygambogic acid;

Gambogyl (4-(2-pyridyl)piperazine);

10-(4-(2-Pyridyl)piperazinyl)gambogyl (4-(2-pyridyl)piperazine);

6-Acetylgambogic acid;

10-(4-(2-Pyridyl)piperazinyl)gambogic acid;

N-Hydroxysuccinimidyl gambogate;

8-(Gambogylamido)octanoic acid;

6-(Gambogylamido)hexanoic acid;

12-(Gambogylamido)dodecanoic acid;

N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;

N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;

N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate;

10-Methoxy-gambogyl piperidine;

Gambogyl (4-(2-pyrimidyl)piperazine);

Gambogyl (bis(2-pyridylmethyl)amine);

Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine);

Gambogyl (4-benzylpiperazine);

Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);

Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine);

Gambogyl (N-methyl-8-(methylamino)-3,6-dioxaoctylamine);

Gambogyl (N-ethyl-2-(ethylamino)ethylamine);

Gambogyl (4-isopropylpiperazine);

Gambogyl (4-cyclopentylpiperazine);

Gambogyl (N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine);

Gambogyl (2,5-dimethylpiperazine);

Gambogyl (3,5-dimethylpiperazine);

Gambogyl (4-(4-acetylphenyl)piperazine);

Gambogyl (4-ethoxycarbonylpiperazine);

Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine);

Gambogyl (4-(2-hydroxyethyl)piperazine);

Gambogyl (N-methyl-2-(methylamino)ethylamine);

Gambogyl (N-methyl-2-(benzylamino)ethylamine);

Gambogyl (N-methyl-(6-methyl-2-pyridyl)methylamine);

Gambogyl (N-ethyl-2-(2-pyridyl)ethylamine);

Gambogyl (N-methyl-(2-pyridyl)methylamine);

Gambogyl (N-methyl-4-(3-pyridyl)butylamine);

Gambogyl (bis(3-pyridylmethyl)amine);

Gambogyl (2,4-dimethyl-2-imidazoline);

Gambogyl (4-methyl-homopiperazine);

Gambogyl (4-(5-hydroxy-3-oxapentyl)piperazine);

Gambogyl (3-dimethylaminopyrrolidine);

Gambogyl ((2-furanyl)methylamine);

Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine);

Gambogyl (3,4,5-trimethoxybenzylamine);

Gambogyl (2-(2-methoxyphenyl)ethylamine);

Gambogyl (2-methoxybenzylamine);

Gambogyl (3,4-methylenedioxybenzylamine);

Gambogyl (2-(2,5-dimethoxyphenyl)ethylamine);

Gambogyl (2-(3-methoxyphenyl)ethylamine);

Gambogyl (3-(piperidinyl)propylamine);

Gambogyl (2-(piperidinyl)ethylamine);

Gambogyl (3,4-dimethoxybenzylamine);

Gambogyl ((2-tetrahydrofuranyl)methylamine);

Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine);

Gambogyl (2-diethylaminoethylamine);

Gambogyl (2,2-dimethyl-3-dimethylaminopropylamine);

Gambogyl ((N-ethoxycarbonyl-4-piperidinyl)amine);

Gambogyl (2-carbamylpyrrolidine);

Gambogyl (3-(homopiperidinyl)propylamine);

Gambogyl ((N-benzyl-4-piperidinyl)amine);

Gambogyl (2-(4-methoxyphenyl)ethylamine);

Gambogyl (4-oxa-hex-5-enylamine);

Ganbogyl (6-hydroxyhexylamine);

Gambogyl (2-(3,5-dimethoxyphenyl)ethylamine);

Gambogyl (3,5-dimethoxybenzylamine); and

Gambogyl (2-carbamyl-2-(4-hydroxyphenyl)ethylamine).

Useful alkyl groups include straight-chained and branched C₁₋₁₀ alkylgroups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,3-pentyl, hexyl and octyl groups, which may be optionally substituted.

Useful alkoxy groups include oxygen substituted by one of the C₁₋₁₀alkyl groups mentioned above, which may be optionally substituted.

Useful alkylthio groups include sulphur substituted by one of the C₁₋₁₀alkyl groups mentioned above, which may be optionally substituted. Alsoincluded are the sulfoxides and sulfones of such alkylthio groups.

Useful amino groups include —NH₂, —NHR₁₁, and —NR₁₁R₁₂, wherein R₁₁ andR₁₂ are C₁₋₁₀ alkyl or cycloalkyl groups, or R₁₁ and R₁₂ are combinedwith the N to form a ring structure, such as a piperidine, or R₁₁ andR₁₂ are combined with the N and another heteroatom to form an optionallysubstituted, saturated or partially saturated 5-7 membered heterocyclogroup, such as a piperazine. The alkyl group may be optionallysubstituted.

Useful heteroatoms include N, O or S.

Optional substituents on the alkyl groups include one or more halo,hydroxy, carboxyl, alkoxycarbonyl, amino, nitro, cyano, C₁-C₆ acylamino,C₁-C₆ aminoacyl, C₁-C₆ acyloxy, C₁-C₆ alkoxy, aryloxy, alkylthio, C₆-C₁₀aryl, C₄-C₇ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, saturated or partiallysaturated 5-7 membered heterocyclo group, or heteroaryl.

Optional substituents on the aryl, aralkyl and heteroaryl groups includeone or more acyl, alkylenedioxy (—OCH₂O—), halo, C₁-C₆ haloalkyl, C₆-C₁₀aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, ureido, cyano,C₁-C₆ acylamino, hydroxy, thiol, C₁-C₆ acyloxy, azido, C₁-C₆ alkoxy, orcarboxy.

Useful heteroalkyl groups contain 1-10 carbon atoms and 1, 2 or 3heteroatoms. Examples of heteroalkyl groups include —CH₂CH₂OCH₂CH₃,—CH₂CH₂OCH₂CH₂OCH₂CH₃, —CH₂CH₂NHCH₃, —CH₂CH₂N(CH₂CH₃)₂,—CH₂CH₂OCH₂CH₂NHCH₃, —CH₂CH₂OCH₂CH₂OCH₂CH₂NHCH₃, —CH₂CH₂NHCH₂CH₃,—CH₂C(CH₃)₂CH₂N(CH₃)₂ or —CH₂(N-ethylpyrrolidine), which may beoptionally substituted.

Optional substituents on heteroalkyl groups include one or more halo,hydroxy, carboxyl, amino, nitro, cyano, alkyl, C₁-C₆ acylamino, C₁-C₆aminoacyl, C₁-C₆ acyloxy, C₁-C₆ alkoxy, aryloxy, alkylthio, C₆-C₁₀ aryl,C₄-C₇ cycloalkyl, C₂-C₆ alkenyl, alkenoxy, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, saturated and unsaturatedheterocyclic, or heteroaryl.

Useful aryl groups are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl. Typical C₆₋₁₄aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.

Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as defined above, as well as cycloalkenyl groups, suchas cyclopentenyl, cycloheptenyl and cyclooctenyl.

Useful halo or halogen groups include fluorine, chlorine, bromine andiodine.

Useful aralkyl groups include any of the above-mentioned C₁₋₁₀ alkylgroups substituted by any of the above-mentioned C₆₋₁₄ aryl groups.Useful values include benzyl, phenethyl and naphthylmethyl.

Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted by one ormore fluorine, chlorine, bromine or iodine atoms, e.g. fluoromethyl,difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl,chloromethyl, chlorofluoromethyl and trichloromethyl groups.

Useful acylamino groups are any C₁₋₆ acyl (alkanoyl) attached to anamino nitrogen, e.g. acetamido, propionamido, butanoylamido,pentanoylamido, hexanoylamido as well as aryl-substituted C₂₋₆substituted acyl groups.

Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to an oxy(—O—) group, e.g. formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy, hexanoyloxy and the like.

Useful saturated or partially saturated 5-7 membered heterocyclo groupsinclude tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl,pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinylpyrazolinyl, tetronoyl and tetramoyl groups.

Optional substitutents on the 5-7 membered heterocyclo groups includeone or more heteroaryl, heterocyclo, alkyl, aralkyl, cycloalkyl,alkoxycarbonyl, carbamyl, aryl or C₁-C₆ aminoacyl.

Useful heteroaryl groups include any one of the following: thienyl,benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furanyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl, 2H-pyrrolyl,pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl,purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g. a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxideand the like.

Optional substituents on the heteroaryl groups include one or moreheteroaryl, heterocyclo, alkyl, aralkyl, cycloalkyl, alkoxycarbonyl,carbamyl, aryl and C₁-C₆ aminoacyl.

Certain compounds of the present invention may exist as stereoisomersincluding optical isomers. The invention includes all stereoisomers andboth the racemic mixtures of such stereoisomers as well as theindividual enantiomers that may be separated according to methods thatare well known to those of ordinary skill in the art.

Examples of pharmaceutically acceptable addition salts include inorganicand organic acid addition salts such as hydrochloride, hydrobromide,phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate,mandelate and oxalate; and inorganic and organic base addition saltswith bases such as sodium hydroxy, Tris(hydroxymethyl)aminomethane(TRIS, tromethane) and N-methyl-glucamine.

Examples of prodrugs of the compounds of the invention include thesimple esters of carboxylic acid containing compounds (e.g. thoseobtained by condensation with a C₁₋₄ alcohol according to methods knownin the art); esters of hydroxy containing compounds (e.g. those obtainedby condensation with a C₁₋₄ carboxylic acid, C₃₋₆ dioic acid oranhydride thereof, such as succinic and fumaric anhydrides according tomethods known in the art); imines of amino containing compounds (e.g.those obtained by condensation with a C₁₋₄ aldehyde or ketone accordingto methods known in the art); and acetals and ketals of alcoholcontaining compounds (e.g. those obtained by condensation withchloromethyl methyl ether or chloromethyl ethyl ether according tomethods known in the art).

The compounds of this invention may be prepared and purified usingmethods known to those skilled in the art, or the novel methods of thisinvention. Specifically, gambogic acid can be purified by 1) preparationof the pyridine salt of the crude extract from gamboge (resin fromGarcinia hanburyi Hook) followed by repeated recrystallization of thesalt in ethanol or 2) converting the salt to the free acid. Using thisprocedure, about 10% by weight of gambogic acid with purity >99% (HPLC)can be obtained from the crude extract. Gambogic acid and analogs ofgambogic acid with Formula I-III also can be separated and purified fromgamboge by repeated chromatography (SiO₂, hexane-EtOAc gradient) using aCombi Flash SG 100 separation system.

Derivatives of gambogic acid with Formula I can be prepared asillustrated by exemplary reactions in Schemes 1 and 2. Reaction ofgambogic acid with methanol in the presence of DMAP and EDC produced themethyl ester of gambogic acid (Scheme 1). Reaction of gambogic acid withpiperidine in the presence of DMAP and EDC produced the piperidinylamide of gambogic acid (Scheme 2).

Derivatives of gambogic acid with Formula I can also be prepared asillustrated by exemplary reactions in Schemes 3-5. Reaction of methylgambogate with methyl iodide in the presence of a base, such as K₂CO₃,produced the methyl-6-methoxy-gambogate (Scheme 3). Reaction of gambogicacid with acetic anhydride in pyridine produced 6-acetyl gambogic acid(Scheme 4). Reaction of gambogic acid with H₂O₂ under basic conditionsproduced 9,10-epoxygambogic acid (Scheme 5).

Derivatives of gambogic acid with Formula II can be prepared asillustrated by exemplary reactions in Schemes 6-10. Reaction of gambogylpiperidine with sodium methoxide produced the methoxy addition productof the amide (Scheme 6). Similarly, reaction of gambogic acid with anamine, such as morpholine, with or without the presence of a base, suchas Et₃N, produced the morpholine addition product of gambogic acid(Scheme 7). Reaction of the piperidine amide of gambogic acid withN-methylpiperazine produced the N-methylpiperazine addition product ofthe amide (Scheme 8). Reduction of gambogic acid by NaBH₄ gave9,10-dihydro-12-hydroxygambogic acid, which may be oxidized byDess-Martin reagent to gave 9,10-dihydro-gambogic acid (Scheme 9).Alternatively, selective reduction of gambogic acid by L-selectride alsoproduced 9,10-dihydrogambogic acid (Scheme 9). Reaction of gambogic acidwith an alkylcuprate, such as cyclohexylcuprate, resulted in theaddition of the alkyl group to the 10-position, thereby producing10-cyclohexyl-gambogic acid (Scheme 10).

An important aspect of the present invention is the discovery thatcompounds having Formula I-III are activators of caspases and inducersof apoptosis. Therefore, these compounds are expected to be useful in avariety of clinical conditions in which there is uncontrolled cellgrowth and spread of abnormal cells, such as in the case of cancer.

Another important aspect of the present invention is the discovery thatcompounds having Formula I-III are potent and highly efficaciousactivators of caspases and inducers of apoptosis in drug resistantcancer cells, such as breast and prostate cancer cells, which enablesthese compounds to kill these drug resistant cancer cells. Incomparison, most standard anti-cancer drugs are not effective in killingdrug resistant cancer cells under the same conditions. Therefore,gambogic acid, its derivatives and analogs are expected to be useful forthe treatment of drug resistant cancer in animals.

The present invention includes a therapeutic method useful to modulatein vivo apoptosis or in vivo neoplastic disease, comprisingadministering to a subject in need of such treatment an effective amountof a compound, or a pharmaceutically acceptable salt or prodrug of acompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis.

The present invention also includes a therapeutic method comprisingadministering to an animal an effective amount of a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-III, wherein said therapeutic method is useful to treat cancer, whichis a group of diseases characterized by the uncontrolled growth andspread of abnormal cells. Such diseases include, but are not limited to,Hodgkin's disease, non-Hodgkin's lymphomas, acute and chroniclymphocytic leukemias, multiple myeloma, neuroblastoma, breastcarcinomas, ovarian carcinomas, lung carcinomas, Wilms' tumor, cervicalcarcinomas, testicular carcinomas, soft-tissue sarcomas, chroniclymphocytic leukemia, primary macroglobulinemia, bladder carcinomas,chronic granulocytic leukemia, primary brain carcinomas, malignantmelanoma, small-cell lung carcinomas, stomach carcinomas, coloncarcinomas, malignant pancreatic insulinoma, malignant carcinoidcarcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides,head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas,acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroidcarcinomas, esophageal carcinomas, malignant hypercalcemia, cervicalhyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemiavera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer,and prostatic carcinomas.

In practicing the therapeutic methods, effective amounts of compositionscontaining therapeutically effective concentrations of the compoundsformulated for oral, intravenous, local and topical application, for thetreatment of neoplastic diseases and other diseases in which caspasecascade mediated physiological responses are implicated, areadministered to an individual exhibiting the symptoms of one or more ofthese disorders. The amounts are effective to ameliorate or eliminateone or more symptoms of the disorders. An effective amount of a compoundfor treating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce, the symptoms associated with thedisease. Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective. The amountmay cure the disease but, typically, is administered in order toameliorate the disease. Typically, repeated administration is requiredto achieve the desired amelioration of symptoms.

In another embodiment, a pharmaceutical composition comprising acompound, or a pharmaceutically acceptable salt of said compound ofFormulae I-III, which functions as a caspase cascade activator andinducer of apoptosis in combination with a pharmaceutically acceptablevehicle is provided.

Another embodiment of the present invention is directed to a compositioneffective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-III, which functions as a caspase cascade activator and inducer ofapoptosis, in combination with at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent. Examples of known anti-cancer agents which can be used forcombination therapy include, but are not limited to, alkylating agentssuch as busulfan, cis-platin, mitomycin C, and carboplatin; antimitoticagents such as colchicine, vinblastine, paclitaxel, and docetaxel; topoI inhibitors such as camptothecin and topotecan; topo II inhibitors suchas doxorubicin and etoposide; RNA/DNA antimetabolites such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites suchas 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;antibodies such as Herceptin® (trastuzumab) and Rituxan® (rituximab).Other known anti-cancer agents which can be used for combination therapyinclude melphalan, chlorambucil, cyclophosamide, ifosfamide,vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin,mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid,tamoxifen and alanosine.

In practicing the methods of the present invention, the compound of theinvention may be administered together with at least one knownchemotherapeutic agent as part of a unitary pharmaceutical composition.Alternatively, the compound of the invention may be administered apartfrom the at least one known cancer chemotherapeutic agent. In thisembodiment, the compound of the invention and the at least one knowncancer chemotherapeutic agent are administered substantiallysimultaneously, i.e. the compounds are administered at the same time orone after the other, so long as the compounds reach therapeutic levelsin the blood.

Another embodiment of the present invention is directed to a compositioneffective to inhibit neoplasia comprising a bioconjugate of saidcompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, in bioconjugation with at least oneknown therapeutically useful antibody, such as Herceptin® (trastuzumab)or Rituxan® (rituximab), growth factors such as DGF, NGF, cytokines suchas IL-2, IL-4, or any molecule that binds to the cell surface. Theantibodies and other molecules will deliver the compound of FormulaeI-III to its target and make it an effective anticancer agent. Thebioconjugate could also enhance the anticancer effect of therapeuticallyuseful antibodies, such as Herceptin® (trastuxumab) or Rituxan®(rituximab).

Similarly, another embodiment of the present invention is directed to acomposition effective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-III, which functions as a caspase cascade activator and inducer ofapoptosis, in combination with radiation therapy. In this embodiment,the compound of the invention may be administered at the same time asthe radiation therapy is administered or at a different time.

Yet another embodiment of the present invention is directed to acomposition effective for post-surgical treatment of cancer, comprisinga compound, or a pharmaceutically acceptable salt or prodrug of saidcompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis. The invention also relates to amethod of treating cancer by surgically removing the cancer and thentreating the animal with one of the pharmaceutical compositionsdescribed herein.

A wide range of immune mechanisms operate rapidly following exposure toan infectious agent. Depending on the type of infection, rapid clonalexpansion of the T and B lymphocytes occurs to combat the infection.

The elimination of the effector cells following an infection is one ofthe major mechanisms maintaining immune homeostasis. This deletion ofreactive cells has been shown to be regulated by a phenomenon known asapoptosis. Autoimnmune diseases have been recently identified to occuras a consequence of deregulated cell death. In certain autoimmunediseases, the immune system directs its powerful cytotoxic effectormechanisms against specialized cells such as oligodendrocytes inmultiple sclerosis, the beta cells of the pancreas in diabetes mellitus,and thyrocytes in Hashimoto's thyroiditis (Ohsako, S. & Elkon, K. B.,Cell Death Differ 6(1):13-21 (1999)). Mutations of the gene encoding thelymphocyte apoptosis receptor Fas/APO-1/CD95 are reportedly associatedwith defective lymphocyte apoptosis and autoimmune lymphoproliferativesyndrome (ALPS), which is characterized by chronic, histologicallybenign splenomegaly and generalized lymphadenopathy,hypergammaglobulinemia, and autoantibody formation. (Infante, A. J., etal., J. Pediatr. 133(5):629-633 (1998) and Vaishnaw, A. K., et al., J.Clin. Invest. 103(3):355-363 (1999)). It was reported thatoverexpression of Bcl-2, which is a member of the Bcl-2 gene family ofprogrammed cell death regulators with anti-apoptotic activity indeveloping B cells of transgenic mice, in the presence of T celldependent costimulatory signals, results in the generation of a modifiedB cell repertoire and in the production of pathogenic autoantibodies(Lopez-Hoyos, M., et al., Int. J. Mol. Med. 1(2):475-483 (1998)). It istherefore evident that many types of autoimmune diseases are caused bydefects of the apoptotic process. One treatment strategy for autoimmunediseases is to turn on apoptosis in the lymphocytes that are causing theautoimnmune disease (O'Reilly, L. A. & Strasser, A., Inflamm Res48(1):5-21 (1999)).

Fas-Fas ligand (FasL) interaction is known to be required for themaintenance of immune homeostasis. Experimental autoimmune thyroiditis(EAT), characterized by autoreactive T and B cell responses and a markedlymphocytic infiltration of the thyroid, is a good model for the studyof the therapeutic effects of FasL. Batteux, F., et al., J. Immunol.162(1):603-608 (1999)) reported that the direct injection of DNAexpression vectors encoding FasL into the inflammed thyroid inhibitedthe development of lymphocytic infiltration of the thyroid. In addition,the death of infiltrating T cells was observed. These results show thatFasL expression on thyrocytes may have a curative effect on ongoing EATby inducing death of pathogenic autoreactive infiltrating T lymphocytes.

Bisindolylmaleimide VIII is known to potentiate Fas-mediated apoptosisin human astrocytoma 1321N1 cells and in Molt-4T cells, and both ofwhich were resistant to apoptosis induced by anti-Fas antibody in theabsence of bisindolylmaleimide VIII. Potentiation of Fas-mediatedapoptosis by bisindolylmaleimide VIII was reported to be selective foractivated, rather than non-activated, T cells, and was Fas-dependent.Zhou T. et al. (Nat Med 5(1):42-8 (1999)) reported that administrationof bisindolylmaleimide VIII to rats during autoantigen stimulationprevented the development of symptoms of T cell-mediated autoimmunediseases in two models: the Lewis rat model of experimental allergicencephalitis and the Lewis adjuvant arthritis model. Therefore, theapplication of a Fas-dependent apoptosis enhancer such asbisindolylmaleimide VIII may be therapeutically useful for the moreeffective elimination of detrimental cells and inhibition of Tcell-mediated autoimmune diseases. Therefore, an effective amount of acompound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, should be an effective treatment forautoimmune disease.

Psoriasis is a chronic skin disease which is characterized by scaly redpatches. Psoralen plus ultraviolet A (PUVA) is a widely used andeffective treatment for psoriasis vulgaris. Coven, et al., PhotodermatolPhotoimmunol Photomed 15(1):22-7 (1999), reported that lymphocytestreated with psoralen 8-MOP or TMP plus UVA displayed DNA degradationpatterns typical of apoptotic cell death. Ozawa, et al., J. Exp. Med189(4):711-718 (1999) reported that induction of T cell apoptosis couldbe the main mechanism by which 312-nm UVB resolves psoriasis skinlesions. Low doses of methotrexate may be used to treat psoriasis torestore a clinically normal skin. Heenen, et al., Arch. Dermatol. Res.290(5):240-245 (1998), reported that low doses of methotrexate mayinduce apoptosis and this mode of action could explain the reduction inepidermal hyperplasia during treatment of psoriasis with methotrexate.Therefore, an effective amount of a compound, or a pharmaceuticallyacceptable salt or prodrug of the compound of Formulae I-III, whichfunctions as a caspase cascade activator and inducer of apoptosis,should be an effective treatment for psoriasis.

Synovial cell hyperplasia is a characteristic of patients withrheumatoid arthritis (RA). Excessive proliferation of RA synovial cellsas well as defects in synovial cell death may be responsible forsynovial cell hyperplasia. Wakisaka, et al., Clin. Exp. Immunol.114(1):119-28 (1998), found that although RA synovial cells could dievia apoptosis through Fas/FasL pathway, apoptosis of synovial cells wasinhibited by proinflammatory cytokines present within the synovium. Thissuggested that inhibition of apoptosis by the proinflammatory cytokinesmay contribute to the outgrowth of synovial cells, and lead to pannusformation and the destruction of joints in patients with RA. Therefore,an effective amount of a compound, or a pharmaceutically acceptable saltor prodrug of the compound of Formulae I-III, which functions as acaspase cascade activator and inducer of apoptosis, should be aneffective treatment for RA.

There has been an accumulation of convincing evidence that apoptosisplays a major role in promoting resolution of the acute inflammatoryresponse. Neutrophils are constitutively programmed to undergoapoptosis, thus limiting their pro-inflammatory potential and leading torapid, specific, and non-phlogistic recognition by macrophages andsemi-professional phagocytes (Savill, J., J. Leukoc. Biol. 61(4):375-80(1997)). Boirivant, et al., Gastroenterology 116(3):557-65 (1999),reported that lamina propria T cells isolated from areas of inflammationin Croln's disease, ulcerative colitis, and other inflammatory statesmanifest decreased CD2 pathway-induced apoptosis. Moreover, studies ofcells from inflamed Crohn's disease tissue indicate that this defect isaccompanied by elevated Bcl-2 levels. Therefore, an effective amount ofa compound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, should be an effective treatment forinflammation.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompounds may be administered to mammals, e.g. humans, orally at a doseof 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceuticallyacceptable salt thereof, per day, per kg of body weight of the mammalbeing treated for apoptosis-mediated disorders. Preferably, about 0.01to about 10 mg/kg is orally administered to treat or prevent suchdisorders. For intramuscular injection, the dose is generally aboutone-half of the oral dose. For example, a suitable intramuscular dosewould be about 0.0025 to about 25 mg/kg, and most preferably, from about0.01 to about 5 mg/kg. If a known cancer chemotherapeutic agent is alsoadministered, it is administered in an amount with is effective toachieve its intended purpose. The amounts of such known cancerchemotherapeutic agents effective for cancer are well known to those ofordinary skill in the art.

The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound of the invention.The unit dose may be administered one or more times daily as one or moretablets each containing from about 0.1 to about 10, conveniently about0.25 to 50 mg of the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally and which can be used forthe preferred type of administration, such as tablets, dragees, andcapsules, and also preparations which can be administered rectally, suchas suppositories, as well as suitable solutions for administration byinjection or orally, contain from about 0.01 to 99 percent, preferablyfrom about 0.25 to 75 percent of active compound(s), together with theexcipient.

Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular apoptosis inducers of the present invention with asolution of a pharmaceutically acceptable non-toxic acid such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular apoptosis inducers of the present invention with a solutionof a pharmaceutically acceptable non-toxic base such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate,Tris, N-methyl-glucamine and the like.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humansand veterinary animals, although the invention is not intended to be solimited.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usemay be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which may be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules may contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose. binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which may be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400 (thecompounds are soluble in PEG-400). Aqueous injection suspensions maycontain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

In accordance with one aspect of the present invention, compounds of theinvention are employed in topical and parenteral formulations and areused for the treatment of skin cancer.

The topical compositions of this invention are formulated preferably asoils, creams, lotions, ointments and the like, by choice of appropriatecarriers. Suitable carriers include vegetable or mineral oils, whitepetrolatum (white soft paraffin), branched chain fats or oils, animalfats and high molecular weight alcohol (greater than C₁₂). The preferredcarriers are those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers may be employed in thesetopical formulations. Examples of such enhancers are found in U.S. Pat.Nos. 3,989,816 and 4,444,762.

Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil, such as almond oil,is admixed. A typical example of such a cream is one which includesabout 40 parts water, about 20 parts beeswax, about 40 parts mineral oiland about 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil, such as almond oil, with warm softparaffin and allowing the mixture to cool. A typical example of such anointment is one which includes about 30% almond oil and about 70% whitesoft paraffin by weight.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 1 Gambogic Acid

Procedure 1:

Step A. The dry gamboge powder (140 g) was extracted with MeOH (3×600mL) at room temperature for 1 week, after filtration, the solvent wasremoved under reduced pressure, gave crude extract (122 g) as yellowpowder.

Step B. Gambogic acid pyridine salt. The above crude extract (120 g) wasdissolved in pyridine (120 mL), then warm water (30 mL) was added to thestirred solution. After cooling to r.t., some precipitate was observed.Hexane (120 mL) was added to the mixture and the mixture was filteredand the solid was washed with hexane and dried. The salt was purified byrepeated recrystallization from ethanol and gave gambogic acid pyridinesalt (7.5 g); HPLC: 99%.

Step C. Gambogic acid. The gambogic acid pyridine salt (0.4 g) wasdissolved in ether (25 mL) and shaken with aqeuous HCl (1N, 25 mL) for 1h. The ether solution was then washed with water (2×10 mL), dried andevaporated to give the title compound (345 mg); HPLC: 99%. ¹H NMR(CDCl₃): 12.66 (s, 1H), 7.43 (d, J=6.9 Hz, 1H), 6.48 (d, J=10.2 Hz, 1H),5.97 (t, J=7.5 Hz, 1H), 5.26 (d, J=9.9 Hz, 1H), 4.91 (m, 2H), 3.37 (m,1H), 3.24-2.98 (m, 2H), 2.81 (d, J=6.6 Hz, 1H), 2.41 (d, J=9 Hz, 1H),2.20 (m, J₁=8.4 Hz, J₂=5.1 HZ, 1H), 1.88 (m, 1H), 1.63 (s, 3H), 1.60 (s,3H), 1.58 (s, 3H), 1.53 (s, 3H), 1.51 (s, 3H), 1.43 (s, 3H), 1.26 (s,3H), 1.18 (s, 3H). MS: 627 (M−H).

Procedure 2:

The crude extract of gamboge (300 mg) was purified by repeated columnchromatography (SiO₂, hexane-EtOAc gradient) using a Combi Flash SG 100separation system, gave 18 mg of gambogic acid; HPLC: 94%, MS. 627(M−H).

EXAMPLE 2 Gambogenic Acid

The crude extract of gamboge (300 mg) was purified as described inExample 1, procedure 2, to give 3 mg of gambogenic acid; HPLC: 84%, MS.629 (M−H).

EXAMPLE 3 Gambogenin

The crude extract of gamboge (300 mg) was purified as described inExample 1, procedure 2, to give 2 mg of gambogenin, HPLC: 71%. MS. 613(M−H).

EXAMPLE 4 Methyl Gambogate

A mixture of gambogic acid (200 mg, 0.32 mmol), DMAP (78 mg, 0.64 mmol),EDC (123 mg, 0.64 mmol) and methanol (102 mg, 3.2 mmol) in THF (5 mL)was stirred at room temperature for 3 h. The solution was poured intowater (10 mL) and was extracted with ethyl acetate (3×10 mL). Thecombined organic layer was dried and concentrated to give the crudeproduct, which was purified by chromatography (SiO₂, EtOAc/Hexane 1:5)to give the title compound (196 mg, 95%). ¹H NMR (CDCl₃): 12.85 (s, 1H),7.54 (d, J=6.9 Hz, 1H), 6.67 (d, J=10.5 Hz, 1H), 5.94 (t, J=6 Hz, 1H),5.43 (d, J=10.2 Hz, 1H), 5.05 (m, 2H), 3.49 (m, 1H), 3.43 (s, 3H),3.35-3.10 (s, 2H), 3.00 (t, J=7.2 Hz, 1H), 2.52 (d, J=10.2 Hz, 1H), 2.32(quar, J1=4.8 Hz, 1H), 2.02 (m, 1H), 1.74 (s, 3H), 1.69 (s, 3H),1.67-1.64 (m, 9H), 1.55 (s, 3H), 1.44 (s, 3H), 1.29 (s, 3H).

EXAMPLE 5 Gambogyl Piperidine

A mixture of gambogic acid (200 mg, 0.32 mmol), DMAP (39 mg, 0.32 mmol),EDC (123 mg, 0.64 mmol) and piperidine (54.2 mg, 0.64 mmol) in THF (3mL) was stirred at room temperature for 6 h. The solution was pouredinto water (10 mL) and was extracted with ethyl acetate (3×10 mL). Thecombined organic layer was dried and concentrated to give the crudeproduct, which was purified by chromatography (SiO₂, EtOAc/CH₂Cl₂ 1:8)to give the title compound (187 mg, 84%). ¹H NMR (CDCl₃): 12.87 (s, 1H),7.53 (d, J=6.9 Hz, 1H), 6.68 (d, J=10.2 Hz, 1H), 5.43 (d, J=10.5 Hz,1H), 5.40 (t, J=6 Hz, 1H), 5.05 (m, 2H), 3.54-3.33 (m, 2H), 3.28 (d,J=6.9 Hz, 1H), 3.11 (t, J=8.4 Hz, 1H), 2.50(d, J=9.6 Hz, 1H), 2.46-2.17(m, 3H), 2.00 (m, 1H), 1.75-1.72 (m, 5H), 1.68 (s, 2H), 1.65 (bs, 6H),1.58 (s, 3H), 1.56 (s, 3H), 1.43 (s, 3H), 1.25 (s, 3H).

EXAMPLE 6 Methyl-6-methoxy-gambogate

A mixture of methyl gambogate (70 mg, 0.11 mmol), anhydrous K₂CO₃ (0.5g), methyl iodide (1 mL) in acetone (5 mL) was stirred at roomtemperature for 70 h. After evaporation to near dryness, water (30 mL)was added into the mixture and it was extracted with ethyl acetate (3×10mL). The combined organic layer was dried and concentrated to give thecrude product, which was purified by chromatography (SiO₂, EtOAc/Hexane1:4) to give the title compound (69 mg, 96%). MS. 657(M+H), 679 (M+Na⁺).¹H NMR (CDCl₃): 7.41 (d, J=6.9 Hz, 1H), 6.64 (d, J=9.9 Hz, 1H), 5.93 (m,J1=6.9 Hz, J2=0.9 Hz, 1H), 5.52 (d, J=10.2 Hz, 1H), 5.05 (m, 2H), 3.79(s, 3H), 3.40 (s, 3H), 3.45-3.18 (m, 3H), 2.95 (d, J=9.6 Hz, 1H), 2.26(m, 1H), 2.02 (m, 1H), 1.73 (s, 3H), 1.66 (d, 3H), 1.63 (bs, 6H), 1.52(s, 3H), 1.42 (s, 3H), 1.27(s, 3H).

EXAMPLE 7 6-Methoxy-gambogyl Piperidine

The title compound was prepared by a procedure similar to that ofExample 6 from gambogyl piperidine and methyl iodide. MS: 710 (M+H), 732(M+Na⁺). ¹H NMR (CDCl₃): 7.40 (d, J=6.9 Hz, 1H), 6.64 (d, J=10.2 Hz,1H), 5.53 (d, J=10.2 Hz, 1H), 5.34 (m, 1H), 5.09 (t, 1H), 5.04 (t, 1H),3.80 (s, 3H), 3.52(m, 3H), 3.38-3.31 (m, 3H), 3.11 (t, 2H), 2.50-1.98(m, 5H), 1.73 (s, 3H), 1.70 (d, 3H), 1.65 (s, 6H), 1.63 (bs, 6H), 1.53(s, 3H), 1.42 (s, 3H), 1.22 (s, 3H).

EXAMPLE 8 10-Morpholinyl-gambogyl Morpholine

A mixture of gambogic acid (100 mg, 0.16 mmol), DMAP (20 mg, 0.16 mmol),EDC (67.4 mg, 0.35 mmol) and morpholine (30.6 mg, 0.35 mmol) in THF (3mL) was stirred at room temperature for 6 h. The solution was pouredinto water (10 mL) and was extracted with ethyl acetate (3×10 mL). Thecombined organic layer was dried and concentrated to give the crudeproduct, which was purified by chromatography (SiO₂, EtOAc/CH₂Cl₂ 1:1)to give the title compound (87 mg, 69%). MS: 785 (M+H), 807 (M+Na⁺). ¹HNMR (CDCl₃): 11.94 (s, 1H), 6.63 (d, J=7.5 Hz, 1H), 5.96 (m, 1H), 5.43(d, J=10.2 Hz, 1H), 5.08 (m, 2H), 3.80 (m, 1H), 3.70-3.12 (m, 12H),2.80-2.36 (m, 7H), 2.05 (m, 1H), 1.95 (m, 1H), 1.87 (s, 3H), 1.73 (bs,3H), 1.64 (bs, 6H), 1.55 (s, 3H), 1.45 (m, 1H), 1.32 (s, 3H), 1.28 (s,3H), 1.24 (3H), 1.07 (s, 3H).

EXAMPLE 9 Gambogyl (4-Methylpiperazine)

A mixture of gambogic acid (93 mg, 0.15 mmol), DMAP (22 mg, 0.18 mmol),EDC (34 mg, 0.18 mmol) and N-methyl piperazine (15 mg, 0.15 mmol) in THF(5 mL) was stirred at room temperature for 5 h. The solution was pouredinto water (50 mL) and was extracted with ethyl acetate (3×10 mL). Thecombined organic layer was dried and concentrated to give crude product,which was purified by chromatography (SiO₂, EtOAc/MeOH 12:1) to give thetitle compound (35 mg, 33%). MS: 709 (M−H), 711 (M+H), 733 (M+Na⁺). ¹HNMR (CDCl₃): 12.85 (s, 1H), 7.52 (d, J=6.6 Hz, 1H), 6.66 (d, J=9.9 Hz,1H), 5.42 (t, J=10.5 Hz, 1H), 5.05 (m, 2H), 3.62 (m, 1H), 3.40 (m, 2H),3.28-3.17 (m, 4H), 2.50-1.98 (m, 7H), 2.23 (s, 3H), 1.72 (bs, 6H), 1.63(bs, 6H), 1.53 (bs, 6H), 1.41 (s, 3H), 1.23 (s, 3H).

EXAMPLE 10 10-Morpholinyl-gambogyl Piperidine

A solution of gambogyl piperidine (50 mg, 0.071 mmol) and morpholine(0.3 mL) in THF (3 mL) was stirred for 48 h. It was evaporated and thecrude product was purified through chromatography to yield the titlecompound (48 mg, 86%). ¹H NMR (CDCl₃) 11.94 (s, 1H), 6.66 (d, J=9.9 Hz,1H), 5.92 (t, 1H), 5.44 (d, J=10.2 Hz, 1H), 5.06 (m, 1H), 3.72-3.12 (m,12H), 2.80 (m, 3H), 2.60-2.40 (m, 4H), 2.06 (m, 2H), 1.88 (s, 3H), 1.75(s, 3H), 1.66 (s, 3H), 1.65 (s, 3H), 1.57 (s, 3H), 1.55 (m, 2H), 1.34(s, 3H), 1.32 (s, 3H), 1.22 (s, 2H), 1.11 (s, 3H).

EXAMPLE 11 10-(4-Metliylpiperazinyl)-gambogyl Piperidine

The title compound was prepared from gambogyl piperidine andN-methylpiperazine by a procedure similar to that of Example 10. MS. 797(M+H), 819 (M+Na), 835 (M+K), 795 (M−H). ¹H NMR (CDCl₃) 12.01 (s, 1H),6.66 (d, J=9.9 Hz, 1H), 5.94 (t, 1H), 5.44 (d, J=10.2 Hz, 1H), 5.12-5.10(m, 2H), 3.80 (d, 1H), 3.52 (d, 1H), 3.38-3.12 (m, 5H), 2.78-2.26 (m,6H), 2.24 (s, 3H), 2.12-2.04 (m, 2H),), 1.89 (s, 3H), 1.75 (s, 3H), 1.66(s, 6H), 1.57 (s, 3H), 1.55 (m, 2H), 1.34 (s, 3H), 1.32 (s, 3H), 1.11(s, 3H).

EXAMPLE 12 N-(2-Gambogylamidoethyl)biotinamide

The title compound was prepared by a procedure similar to that ofExample 9 from gambogic acid and N-(2-aminoethyl)biotinamide. MS: 919(M+Na), 897 (M+H), 895 (M−H). ¹H NMR (CDCl₃): 12.9, 12.78 (1H),7.60-7.57 (m, 1H), 6.90 (m, 1H), 6.78 (m, 1H), 6.70-6.62 (m, 1H),5.48(d, J=9.9 Hz, 1H), 5.42 (m, 1H), 5.30 (m, 1H), 5.08 (m, 2H), 4.66(s,1H), 4.49 (m, 1H), 4.33 (m, 1H), 3.58-3.40 (m, 2H), 3.38-3.10 (m,5H), 3.16-2.88 (m, 1H), 2.80-2.52 (m, 2H), 2,40-1.92 (m, 6H), 1.78 (bs,3H), 1.74 (bs, 2H), 1.73 (bs, 3H), 1.69 (bs, 3H), 1.65 (bs, 6H), 1.55(bs, 3H), 1.50-1.20 (m, 13H), 1.2-0.88 (m, 4H).

EXAMPLE 13 10-(4-Methylpiperazinyl)gambogic Acid

A solution of gambogic acid (35 mg, 0.056 mmol) and N-methylpiperazine(0.5 mL) in THF (4 mL) was stirred for 24 h, then another portion ofN-methylpiperazine (0.5 mL) was added and stirred for 48 h. The solutionwas diluted with EtOAc (30 mL) and washed with aqueous NH₄Cl (3×30 mL).After concentration, the mixture was dissolved in ethyl ether (15 mL)and washed with 0.1 N HCl. After concentration, the residue was washedwith hexane four times to gave the title compound (9 mg, 20 %). ¹H NMR(CDCl₃) 11.8 (s, 1H), 6.64 (d, J=9.9 Hz, 1H), 6.57(t, 1H), 5.45 (d,J=10.5 Hz, 1H), 5.09 (t, 1H), 3.51 (bs, 1H), 3.30-2.70 (m, 11H), 2.81(s, 3H), 2.51 (d, J=8.4 Hz, 2H), 2.12-2.02 (m, 2H), 1.96 (s, 3H), 1.73(s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), 1.35 (s, 6H), 1.26(m, 2H), 1.11 (s, 3H), 0.88 (m, 2H).

EXAMPLE 14 10-Piperidyl-gambogyl Piperidine

A mixture of gambogic acid (460 mg, 0.73 mmol), EDCI (166 mg, 0.87mmol), DMAP (47 mg, 0.38 mmol) and piperidine (75 μL, 0.76 mmol) in THF(5 mL) was stirred at room temperature for 40 h. It was diluted with 1:1hexane/EtOAc (80 mL), washed with water and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (3:2hexane/EtOAc) to yield the title compound as a pale yellow solid (40 mg,0.051 mmol, 7%) and gambogyl 1′-piperidine (220 mg, 0.32 mmol). ¹H NMR(CDCl₃): 12.05 (s, 1H), 6.66 (d, J=10.0 Hz, 1H), 5.95 (t, J=6.3 Hz, 1H),5.44 (d, J=10.0 Hz, 1H), 5.12-5.03 (m, 2H), 3.75-3.10 (m, 9H), 2.76-2.66(m, 3H), 2.49 (d, J=8.4, 2H), 2.34(m, 2H), 2.08 (m, 3H), 1.89 (s, 3H),1.75 (s, 3H), 1.66 (d, 3H), 1.61-1.23 (m, 15H), 1.33 (s, 3H), 1.31 (s,3H), 1.27(s, 3H), 1.26 (s, 3H).

EXAMPLE 15 10-Piperidinyl-gambogic Acid

Gambogic acid (10 mg, 0.016 mmol) in piperidine (0.5 mL) was stirred atroom temperature for 40 h. The solvent was removed in vacuo. The residuewas diluted with 1:2 hexane/EtOAc (50 mL), washed with saturatedammonium chloride aqueous solution followed by brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by chromatography(3:2 hexane/EtOAc) to yield one of the diasteromers of the titlecompound (A, 3 mg, 0.004 mmol, 26%) and the other diastereomer (B, 1 mg,0.001 mmol, 6%). ¹H NMR (CDCl₃): diastereomer A: 12.00 (s, 1H), 6.66 (d,J=9.9 Hz, 1H), 6.52 (t, J=6.9 Hz, 1H), 5.46 (d, J=9.9 Hz, 1H), 5.12-5.05(m, 2H), 3.32-3.04 (m, 6H), 2.81 (t, J=4.5, 1H), 2.55-2.43 (m, 3H),2.33(m, 2H), 2.12-1.91 (m, 3H), 1.98 (s, 3H), 1.74 (s, 3H), 1.66 (s,3H), 1.63 (s, 3H), 1.35 (s, 6H), 1.50-1.28 (m, 6H), 1.14 (s, 3H);diastereomer B: 12.00 (s, 1H), 7.37 (t, J=6.3 Hz, 1H), 6.66 (d, J=10.0Hz, 1H), 5.46 (d, J=10.0 Hz, 1H), 5.12-5.02 (m, 2H), 3.35-3.18 (m, 3H),3.11 (s, 1H), 2.91-2.79 (m, 3H), 2.56-2.48 (m, 3H), 2.33(m, 2H),2.12-1.94 (m, 5H), 1.87 (s, 3H), 1.74 (s, 3H), 1.66 (s, 3H), 1.63 (s,3H), 1.40 (s, 3H), 1.34 (s, 3H), 1.50-1.28 (m, 6H), 1.13 (s, 3H).

EXAMPLE 16 9,10-Dihydro-12-hydroxygambogic Acid

To a solution of gambogic acid (14 mg, 0.022 mmol) in methanol (2 mL)was added NaBH₄ (22 mg, 0.58 mmol) at 0° C. The mixture was stirred for3 h and the cooling bath was allowed to slowly warm to room temperature.Acetone (0.5 mL) was added to the mixture and it was stirred for 30min., acidified with 2 N HCl to pH 6, diluted with EtOAc (40 mL), washedwith water (3 times) and brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by chromatography (9:1 EtOAc/MeOH) togive the title compound as an oil (9 mg, 0.014 mmol, 66%). ¹H NMR(CDCl₃): 12.02 (s, 1H), 6.66 (d, J=10.2 Hz, 1H), 6.33 (t, J=7.2, 1H),5.45 (d, J=10.2 Hz, 1H), 5.12-5.05 (m, 2H), 3.71 (s, 1H), 3.26-3.11 (m,3H), 3.02-2.94 (m, 2H), 2.56-2.49 (m, 1H), 2.36 (d, J=9.6, 1H),2.12-2.04 (m, 3H), 1.99 (s, 3H), 1.76 (m, 1H), 1.73 (s, 3H), 1.66 (s,3H), 1.63 (s, 3H), 1.60-1.30 (m, 5H), 1.42 (s, 3H), 1.39 (s, 3H), 1.35(s, 3H).

EXAMPLE 17 Methyl-10-Morpholinyl-gambogate

To a solution of methyl gambogate (50 mg, 0.078 mmol) in THF (2 mL) wasadded morpholine (70 L, 0.80 mmol). The mixture was stirred at roomtemperature for 17 h, diluted with 1:1 hexane/EtOAc (100 mL), washedwith water (3 times) and brine, dried over Na₂SO₄ and concentrated invacuo to yield the title compound as light yellow solid (52 mg, 0.071mmol, 91%). ¹H NMR (CDCl₃): 11.98 (s, 1H), 6.66 (d, J=10.2 Hz, 1H), 6.62(t, J=6.6 Hz, 1H), 5.46 (d, J=10.2 Hz, 1H), 5.12-5.00 (m, 2H), 3.68 (s,3H), 3.74-3.55 (m, 4H), 3.43-3.14 (m, 6H), 2.77 (m, 1H), 2.59-2.40 (m,5H), 2.07(m, 1H), 1.95 (s, 3H), 1.74 (s, 3H), 1.66 (s, 3H), 1.63 (s,3H), 1.36 (s, 3H), 1.35 (s, 3H), 1.14 (s, 3H).

EXAMPLE 18 Isogambogic Acid

The crude extract of gamboge (300 mg) was purified as described inExample 1, procedure 2, to give 2 mg of isogambogic acid; MS. 627 (M−H).

EXAMPLE 19 Morellic Acid

The crude extract of gamboge (300 mg) was purified as described inExample 1, procedure 2, to give 2 mg of morellic acid; MS. 559 (M−H).

EXAMPLE 20 10-Cyclohexylgambogic Acid

To a solution of gambogic acid (80 mg, 0.13 mmol) in THF (5 mL) wasadded a solution of cyclohexylcuprate (1.2 mmol) in THF prepared fromcyclohexylmagnesium chloride and CuI at 0° C. The mixture was stirredfor 2 h and the cooling bath was allowed to slowly warm to roomtemperature. The reaction was quenched with 2 N HCl and diluted with 1:1hexane/EtOAc (80 mL). The resulting mixture was washed with water andbrine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by chromatography (3:1 hexane/EtOAc) to give the title compoundas an oily solid (9 mg, 0.013 mmol, 10%). ¹H NMR (CDCl₃): 6.61 (d,J=10.2, 1H), 6.14 (t, J=6.0, 1H), 5.39 (d, J=10.2, 1H), 5.20 (t, J=6.6,1H), 5.06 (t, J=7.2, 1H), 3.64 (m, 1H), 3.35-3.10 (m, 3H), 2.82 (br s,2H), 2.67-2.61 (m, 2H), 1.76(s, 3H), 1.72 (s, 3H), 1.68 (s, 3H), 1.66(s, 3H), 1.56 (s, 3H), 1.44 (s, 3H), 1.94-1.25 (m, 15H).

EXAMPLE 21 10-Methylgambogic Acid

The title compound was prepared by a procedure similar to that ofExample 20 from gambogic acid and methylcuprate and was isolated as anoil. ¹H NMR (CDCl₃): 6.58 (d, J=10.2, 1H), 6.12 (t, J=6.6, 1H), 5.36 (d,J=9.9, 1H), 5.15 (t, J=6.6, 1H), 5.04 (t, J=7.2, 1H), 3.32-3.10 (m, 2H),2.93 (d, J=8.4, 1H), 2.84 (d, J=6.3, 2H), 2.62 (d, J=7.6, 1H), 2.40 (t,J=7.2, 1H), 2.25 (d, J=4.8, 1H), 2.08-1.92 (m, 4H), 1.73(s, 3H), 1.68(s, 3H), 1.66 (s, 6H), 1.64 (s, 3H), 1.54 (s, 3H), 1.41 (s, 3H), 1.35(d, J=6.9, 3H), 1.23 (s, 1H).

EXAMPLE 22 9,10-Dihydrogambogic Acid

To a solution of gambogic acid (17 mg, 0.027 mmol) in methylenechloride(2 mL) was added L-selectride solution in THF (1.0 mL, 0.5 mmol)dropwise at −78° C. After 30 min of stirring, the reaction was quenchedwith 1 mL of 2 N HCl. The mixture was then allowed to warm to roomtemperature and was diluted with 1:1 hexane/EtOAc (50 mL). The resultingmixture was washed with water and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (2:3hexane/EtOAc/) to give the title compound as an oil (0.6 mg, 0.001 mmol,4%). ¹H NMR (CDCl₃): 11.96 (s, 1H), 6.67 (d, J=10.2, 1H), 6.54 (t,J=6.6, 1H), 5.46 (d, J=10.2, 1H), 5.13-5.05 (m, 2H), 3.33-3.16 (m, 3H),2.85 (d, J=13.8, 1H), 2.60 (d, J=8.7, 1H), 2.43 (s, 1H), 2.08 (m, 1H),1.97 (s, 3H), 1.74 (s, 3H), 1.67 (s, 6H), 1.64 (s, 3H), 1.57 (s, 3H),1.37 (s, 3H), 1.36 (d, J=6.9, 3H), 1.31-1.22 (m, 5H), 1.14 (s, 3H).

EXAMPLE 23 Gambogyl (2-(4-Morpholinyl)ethylamine)

The title compound was prepared as described in Example 5 from gambogicacid and 2-(4-morpholinyl)ethylamine and isolated as a yellow solid (75mg, 0.10 mmol, 56%). ¹H NMR (CDCl₃): 12.86 (s, 1H), 7.54 (d, J=6.6, 1H),6.68 (d, J=10.2, 1H), 6.56 (t, J=5.1, 1H), 5.46 (d, J=10.2, 1H), 5.28(d, J=7.5, 1H), 5.05 (br s, 1H), 3.68 (t, J=4.2, 4H), 3.47 (m, 1H),3.71-3.17 (m, 4H), 2.68 (t, J=6.6, 2H), 2.54 (d, J=9.6, 1H), 2.48-2.44(m, 6H), 2.36-2.30 (m, 1H), 2.01-2.00 (m, 3H), 1.74 (s, 6H), 1.67 (s,3H), 1.65 (s, 6H), 1.61 (s, 3H), 1.44 (s, 3H), 1.28 (s, 3H).

EXAMPLE 24 9,10-Epoxygambogic Acid

To a solution of gambogic acid (52 mg, 0.08 mmol) in methanol (2 mL) wasadded 2 N NaOH (0.5 mL, 1.0 mmol), followed by 35% H₂O₂. (0.2 mL, 2.1mmol) at room temperature. The mixture was stirred at room temperaturefor 10 min, diluted with 1:1 hexane/EtOAc (50 mL), washed with water, 2N HCl and brine, dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by chromatography (1:2 hexane/EtOAc) to yield thetitle compound as an oil (2.2 mg, 0.003 mmol, 4%). ¹H NMR (CDCl₃): 11.92(s, 1H), 6.66 (d, J=10.2, 1H), 6.51 (t, J=6.9, 1H), 5.46 (d, J=9.9, 1H),5.09-5.04 (m, 2H), 4.35 (d, J=3.9, 1H), 3.32 (s, 2H), 3.27-2.99 (m, 4H),2.85 (t, J=4.8, 1H), 2.51 (d, J=8.7, 1H), 2.07 (m, 1H), 1.97 (s, 3H),1.74 (s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), 1.36 (s, 3H),1.15 (s, 3H).

EXAMPLE 25 Gambogyl (4-(2-Pyridyl)piperazine) and10-[4-(2-Pyridyl)piperazinyl]gambogyl(4-(2-Pyridyl)piperazine)

A mixture of gambogic acid (230 mg, 0.37 mmol), 1-(2-pyridyl)piperazine(75 μL, 0.46 mmol), and EDC (77 mg, 0.40 mmol) in DMF (3 mL) was stirredat room temperature, overnight. The mixture was diluted with 1:1hexane/EtOAc (90 mL), washed with water and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (3:2hexane/EtOAc) to yield 10 mg of gambogyl (4-(2-pyridyl)piperazine) as ayellow solid. ¹H NMR (CDCl₃): 12.87 (s, 1H), 8.19 (m, 1H), 7.52 (d,J=6.9, 1H), 6.68-6.62 (m, 2H), 6.43 (d, J=9.9, 1H), 5.06 (br s, 2H),3.76-3.27 (m, 11H), 2.52-2.00 (m, 6H), 1.76 (s, 3H), 1.26 (s, 3H); 1.73(s, 3H), 1.68 (s, 3H), 1.65 (s, 6H), 1.56 (s, 3H), 1.42 (s, 3H), 1.26(s, 3H); and 31 mg of10-[4-(2-pyridyl)piperazinyl]gambogyl(4-(2-pyridyl)piperazine) as ayellow solid. ¹H NMR (CDCl₃): 11.99 (s, 1H), 8.17 (d, J=4.8, 2H), 7.45(t, J=7.5, 2H), 6.68-6.52 (m, 5H), 6.00 (t, J=6.6, 1H), 5.44 (d, J=10.2,1H), 5.11-5.07 (m, 2H), 3.90-3.13 (m, 15H), 2.83-2.51 (m, 8H), 2.07 (m,2H), 1.90 (s, 3H), 1.73 (s, 3H), 1.65 (s, 6H), 1.57 (s, 3H), 1.35 (s,3H), 1.32 (s, 3H), 1.12 (s, 3H);

EXAMPLE 26 6-Acetyl-gambogic Acid

A mixture of gambogic acid (154 mg, 0.24 mmol) and Ac₂O (0.3 mL, 3.2mmol) in pyridine (3 mL) was stirred at room temperature for four days.The mixture was diluted with 1:1 hexane/EtOAc (80 mL), washed withwater, 2 N HCl and brine, dried over Na₂SO₄ and concentrated in vacuo.The residue was purified by chromatography (1:2 hexane/EtOAc) to yieldthe title compound as a yellow solid (47 mg, 0.07 mmol, 29%). ¹H NMR(CDCl₃): 7.44 (d, J=6.9, 1H), 6.66 (t, J=6.6, 1H), 6.40 (d, J=10.2, 1H),5.60 (d, J=10.5, 1H), 5.13 (t, J=6.9, 1H), 5.04 (t, J=6.9, 1H), 3.46 (m,1H), 3.34 (d, J=6.9, 1H), 2.67-2.50 (m, 3H), 2.39 (s, 3H), 2.33-2.27 (m,1H), 2.08-1.98 (m, 2H), 1.73 (s, 3H), 1.71 (s, 3H), 1.65 (s, 6H), 1.54(s, 3H), 1.40 (s, 3H), 1.6 (s, 3H), 1.29 (s, 3H).

EXAMPLE 27 10-[4-(2-Pyridyl)piperazinyl]gambogic Acid

A mixture of pyridinium gambogate (228 mg, 0.32 mmol) and1-(2-pyridyl)piperazine (289 mg, 1.8 mmol) in THF (3 mL) was stirred atroom temperature, overnight. The mixture was diluted with 1:1hexane/EtOAc (80 mL), washed with water, 2 N HCl and brine, dried overNa₂SO₄ and concentrated in vacuo to yield the title compound as a yellowsolid (143 mg, 0.16 mmol, 50%). ¹H NMR (CDCl₃): 11.97 (s, 1H), 8.15 (m,1H), 7.45 (m, 1H), 6.68-6.54 (m, 4H), 5.46 (d, J=9.9, 1H), 4.12-5.02 (m,2H), 3.40-3.08 (m, 10H), 2.83 (t, J=4.5, 1H), 2.71-2.67 (m, 2H),2.57-2.52 (m, 3H), 1.95 (s, 3H), 1.74 (s, 3H), 1.66(s, 3H), 1.63 (s,3H), 1.57 (s, 3H), 1.37 (s, 6H), 1.16 (s, 3H).

EXAMPLE 28 N-Hydroxysuccinimidyl Gambogate

A mixture of gambogic acid (600 mg, 0.96 mmol), N-hydroxysuccinimide(221 mg, 1.92 mmol), DCC (296.6 mg, 1.44 mmol) in dichloromethane (20mL) was stirred for 2 h. It was evaporated to dryness and the residuewas dissolved in ethyl acetate (50 mL) and washed with water (50 mL×3).The organic layer was dried and concentrated to give crude product,which was purified by flash column chromatography (SiO₂, EtOAc/hexane1:3) to give the title compound (530 mg, 76%). ¹H NMR (CDCl₃): 12.85 (s,1H), 7.55 (d, J=6.9 Hz, 1H), 6.67 (d, J=10.2 Hz, 1H), 6.62 (t, J=6.9 Hz,1H), 5.44 (d, J=9.9 Hz, 1H), 5.06 (m, 2H), 3.46 (m, 1H), 3.38-3.12 (m,2H), 2.84-2.76 (m, 4H), 2.54 (d, 1H), 2.30 (m, 1H), 2.04 (m, 1H), 1.94(s, 3H), 1.74(s, 3H), 1.72 (s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s,3H), (bs, 6H), 1.43 (s, 3H), 1.29 (s, 3H).

EXAMPLE 29 8-(Gambogylamido)octanoic Acid

A solution of 8-aminooctanoic acid (3.07 mg, 0.019 mmol),N-hydroxysuccinimidyl gambogate (14 mg, 0.019 mmol), triethylamine (0.15mL) in anhydrous DMSO (3 mL) was stirred overnight. It was diluted withwater and extracted with ethyl acetate (3×10 mL). The combined organiclayer was dried and concentrated to give crude product, which waspurified by column chromatography (SiO₂, EtOAc/MeOH 10:1) to give thetitle compound (11 mg, 67%). ¹H NMR (CDCl₃): 12.80 (bs, 1H), 7.58 (bs,1H), 6.64 (d, J=9.3 Hz, 1H), 5.50-5.00 (m, 4H), 3.54 (bs, 1H), 3.32-3.00(m, 3H), 3.50-2.42 (m, 4H), 1.75 (s, 3H), 1.72 (s, 3H), 1.70 (s, 3H).MS. 792 (M+Na⁺), 768 (M−H).

EXAMPLE 30 6-(Gambogylamido)hexanoic Acid

The title compound was prepared by a procedure similar to that ofExample 29. ¹H NMR (CDCl₃): 12.70 (bs, 1H), 7.58 (bs, 1H), 6.62 (bs,1H), 5.40 (bs, 1H), 5.20 (bs, 1H), 5.00 (bs, 2H), 3.60-3.00 (m, 4H),3.50-2.42 (m, 4H), 1.74 (s, 3H), 1.72 (s, 3H), 1.69 (s, 3H). MS. 764(M+Na⁺), 740 (M−H).

EXAMPLE 31 12-(Gambogylamido)dodecanoic Acid

The title compound was prepared by a procedure similar to that ofExample 29. ¹H NMR (CDCl₃): 12.7 (bs, 1H), 7.48 (d, 1H), 6.64 (d, J=10.5Hz, 1H), 5.50-5.00 (m, 6H), 3.50 (bs, 1H), 3.40-3.00 (m, 3H), 2.80-1.92(m, 6H). 1.75 (s, 3H), 1.73 (s, 3H), 1.71 (s, 3H), 1.56 (s, 3H). MS. 849(M+Na⁺), 825 (M−1).

EXAMPLE 32 N-Hydroxysuccinimidyl-8-(Gambogylamido)octanoate

The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.55 and 7.51 (d, J=6.9 Hz,1H), 6.65 and 6.64 (d, J=10.2 Hz, 1H), 5.50-5.00 (m, 4H), 4.12 (d, 2H),3.49 (m, 2H), 3.30 (t, J=6.6 Hz, 1H), 3.19 (m, 3H), 2.85 (s, 4H),2.70-2.50 (m, 3H), 2.04 (m, 1H), 1.75 (bs, 3H), 1.74(s, 3H), 1.72 (s,3H), 1.70 (s, 3H), 1.56 (s, 3H), 1.42 (s, 3H), 1.33 (bs, 3H), 1.30 (bs,3H). MS. 889 (M+Na⁺), 865 (M−H).

EXAMPLE 33 N-Hydroxysuccinimidyl-6-(Gambogylamido)hexanoate

The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.56 and 7.52 (d, J=6.9 Hz,1H), 6.69 and 6.65(d, J=10.2 Hz, 1H), 6.59 (t, 1H), 5.60-5.00 (m, 4H),4.10 (m, 2H), 3.60-3.12 (m, 6H), 2.85 (s, 4H), 2.70-2.50 (m, 3H), 2.35(m, 1H), 2.04 (m, 1H), 1.90 (m, 4H), 1.73(s, 3H), 1.72 (s, 3H), 1.69 (s,3H), 1.56 (bs, 6H), 1.44(s, 3H), 1.33(s, 3H), 1.29(s, 3H). MS. 861(M+Na⁺), 837 (M−H).

EXAMPLE 34 N-Hydroxysuccinimidyl-12-(Gambogylamido)dodecanoate

The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.55 and 7.51 (d, J=7.2 Hz,1H), 6.66 and 6.64(d, J=9.9 Hz, 1H), 5.47(d, J=10.5 Hz, 1H), 5.46-5.10(m, 3H), 4.08 (m, 4H), 3.56-3.40 (m, 4H), 3.18 (m, 2H), 2.60 (t, 1H),2.83 (s, 4H). MS. 946 (M+Na⁺), 922 (M−H).

EXAMPLE 35 10-Methoxy-gambogyl Piperidine

To a solution of gambogyl piperidine (30 mg, 0.043 mmol) in methanol (4mL) was added sodium methoxide (4.6 mg, 0.086 mmol) and it was stirredat room temperature for 3 h. The reaction was poured into ice water (20mL), and extracted with ethyl acetate (3×10 mL). The organic extract wasdried and concentrated to give crude product, which was purified bychromatography to give the title compound (18 mg, 58%). MS. 726 (M−H⁺),750 (M+Na⁺). ¹H NMR (CDCl₃): 11.98 (s, 1H), 6.65 (d, J=10.2 Hz, 1H),5.77 (t, J=6.6 Hz, 1H), 5.43 (d, J=10.2 Hz, 1H), 5.07 (m, 2H), 4.33 (d,1H), 3.60-3.15 (m, 3H), 3.31 (s, 3H), 2.80-2.40 (m, 3H), 1.87 (s, 3H),1.66 (s, 3H), 1.60 (s, 3H), 1.36 (s, 3H), 1.31 (s, 3H), 1.28 (s, 3H),1.23 (s, 3H), 1.11 (s, 3H).

EXAMPLE 36 Gambogyl (2-Dimethylaminoethylamine)

The title compound was prepared by a procedure similar to that ofExample 29. MS. 697 (M−H⁻), 699 (M+H⁺). ¹H NMR (CDCl₃): 12.90 (bs, 1H),7.54 (d, J=6.9 Hz, 1H), 6.68 (d, J=9.6 Hz, 1H), 6.52 (t, 1H), 5.45 (d,J=10.2 Hz, 1H), 5.37 (dt, J₁=8.4 Hz, J₂ =1.5 Hz, 1H), 5.05 (m, 2H),3.50-3.10 (m, 3H), 2.21 (s, 6H), 1.76 (s, 3H), 1.75 (s, 3H), 1.69 (s,3H), 1.65 (s, 3H), 1.64 (s, 3H), 1.56 (s, 3H), 1.44 (s, 3H), 1.29 (s,3H).

The following compounds (Examples 37-89) were prepared by a proceduresimilar to that of Example 9.

TABLE I Examples 37-89 Example # STRUCTURE MF MW 37

C₄₆H₅₄N₄O₇ 774.954 38

C₅₀H₅₅N₃O₇ 809.998 39

C₅₀H₅₄N₂O₈ 810.983 40

C₄₉H₅₈N₂O₇ 787.004 41

C₅₀H₅₈N₂O₉ 831.013 42

C₄₄H₅₈N₂O₈ 742.948 43

C₄₆H₆₂N₂O₉ 787.001 44

C₄₄H₅₈N₂O₇ 726.949 45

C₄₅H₅₈N₂O₇ 738.96 46

C₄₇H₆₀N₂O₇ 764.998 47

C₄₈H₅₆N₂O₉ 804.975 48

C₄₄H₅₆N₂O₇ 724.933 49

C₄₄H₅₆N₂O₇ 724.933 50

C₅₀H₅₈N₂O₈ 815.014 51

C₄₅H₅₆N₂O₉ 768.942 52

C₄₈H₆₁N₃O₈ 808.023 53

C₄₄H₅₆N₂O₈ 740.932 54

C₄₂H₅₄N₂O₇ 698.896 55

C₄₈H₅₈N₂O₇ 774.993 56

C₄₆H₅₄N₂O₇ 746.94 57

C₄₇H₅₆N₂O₇ 760.966 58

C₄₅H₅₂N₂O₇ 732.913 59

C₄₅H₅₈N₂O₇ 774.993 60

C₅₀H₅₅N₃O₇ 809.998 61

C₄₃H₅₂N₂O₇ 708.891 62

C₄₄H₅₆N₂O₇ 724.933 63

C₄₆H₆₀N₂O₉ 784.985 64

C₄₄H₅₆N₂O₇ 724.933 65

C₄₃H₄₉NO₈ 707.859 66

C₄₇H₅NO₈ 761.951 67

C₄₇H₅₅NO₈ 761.951 68

C₄₈H₅₇NO₁₀ 807.975 69

C₄₆H₅₃NO₈ 747.924 70

C₄₆H₅₁NO₉ 761.907 71

C₄₈H₅₇NO₉ 791.976 72

C₄₇H₅₅NO₈ 761.951 73

C₄₆H₆₀N₂O₇ 752.987 74

C₄₅H₅₈N₂O₇ 738.96 75

C₄₇H₅₅NO₉ 777.95 76

C₄₃H₅₃NO₈ 711.891 77

C₄₅H₅₈N₂O₇ 738.96 78

C₄₄H₅₈N₂O₇ 726.949 79

C₄₅H₆₀N₂O₇ 740.976 80

C₄₆H₅₈N₂O₉ 782.969 81

C₄₃H₅₂N₂O₈ 724.89 82

C₄₇H₆₂N₂O₇ 767.014 83

C₅₀H₆₀N₂O₇ 801.031 84

C₄₇H₅₅NO₈ 761.951 85

C₄₃H₅₃NO₈ 711.891 86

C₄₄H₅₇NO₈ 727.933 87

C₄₈H₅₇NO₉ 791.976 88

C₄₇H₅₅NO₉ 777.95 89

C₄₇H₅₄N₂O₉ 790.949

EXAMPLE 90 Identification Of Gambogic Acid And Analogs As AntineoplasticCompounds That are Caspase Cascade Activators

Human breast cancer cell lines T-47D and ZR-75-1, human prostate cancercell line PC-3, human leukemia cancer cell line HL-60 and humannon-transformed fibroblast cell line MRC-5 cells were grown according tomedia component mixtures designated by The American Type CultureCollection+10% FCS (Life Technologies, Inc.), in a 5% CO₂-95% humidityincubator at 37° C. T-47D, ZR-75-1 and PC-3 cells were maintained at acell density between 30 and 80% confluency and for HL-60 at a celldensity of 0.1 to 0.6×10⁶ cells/ml. Cells were harvested at 600×g andresuspended at 0.65×10⁶ cells/ml into appropriate media+10% FCS. Analiquot of 45 μl of cells was added to a well of a 96-well microtiterplate containing 5 μl of a 10% DMSO in RPMI-1640 media solutioncontaining 1.6 to 100 μM gambogic acid or other test compound (0.16 to10 μM final). An aliquot of 45 μl of cells was added to a well of a96-well microtiter plate containing 5 μl of a 10% DMSO in RPMI-1640media solution without test compound as the control sample. The sampleswere mixed by agitation and then incubated at 37° C. for 24 h in a 5%CO₂-95% humidity incubator. After incubation, the samples were removedfrom the incubator and 50 μl of a solution containing 20 μM ofN-(Ac-DEVD)-N′-ethoxycarbonyl-R110 fluorogenic substrate (SEQ ID NO:1)(Cytovia, Inc.; WO99/18856), 20% sucrose (Sigma), 20 mM DTT (Sigma), 200mM NaCl (Sigma), 40 mM Na PIPES buffer pH 7.2 (Sigma), and 500 μg/mllysolecithin (Calbiochem) was added. The samples were mixed by agitationand incubated at room temperature. Using a fluorescent plate reader(Model 1420 Wallac Instruments), an initial reading (T=0) was madeapproximately 1-2 min after addition of the substrate solution,employing excitation at 485 nm and emission at 530 nm, to determine thebackground fluorescence of the control sample. After the 3 h incubation,the samples were read for fluorescence as above (T=3 h).

Calculation:

The Relative Fluorescence Unit values (RFU) were used to calculatesample readings as follows:

RFU_((T=3hr))−Control RFU_((T=0))=Net RFU_((T=3hr))

The activity of caspase cascade activation was determined by the ratioof the net RFU value for gambogic acid or other test compound to that ofcontrol samples. The EC₅₀ (nM) was determined by a sigmoidaldose-response calculation (Prism 2.0, GraphPad Software Inc.). Thecaspase activity (Ratio) potency (EC₅₀) are summarized in Table II:

TABLE II Caspase Activity and Potency T-47D ZR-75-1 PC-3 HL-60 MRC-5Example Ratio Ratio Ratio Ratio Ratio # (nM) ED50 (nM) ED50 (nM) ED50(nM) ED50 (nM) ED50 1 13.6 560 11.9 1400 2.1 1500 6.3 400 17.8 1410 416.8 484 14.9 1640 3.9 1330 7.7 339 27.8  501 5 13.9 210 14.3  783 2.7 900 5.2 200 12.6  631 6 12.0 2800  ND ND 4.5 5000 ND ND ND ND 2 16.9310 14.2 1160 2.9 1350 5.6 340 22.4 1260 3  7.0 1000  ND ND 2.9 1700 NDND ND ND 7 14.4 830 13.4 1650 2.3 1700 ND ND  9.4 1200 9 11.7 990 12.82050 3.1 5900 ND ND 11.4 1900 ND = not determined

Thus, gambogic acid and its derivatives and analogs are identified aspotent caspase cascade activators and antineoplastic compounds in thisassay.

EXAMPLE 91 Identification Of Gambogic Acid And Analogs As AntineoplasticCompounds That Exhibit Inhibition Of Cell Proliferation (GI₅₀) And CellDeath (LC₅₀)

T-47D, ZR-75-1, PC-3, human prostate cancer cell line DU-145, humannon-small cell lung cancer cell line A-549, human small cell lung cancercell line SHP-77, HL-60 and MRC-5 cells were grown and harvested as inExample 90. An aliquot of 90 μl of cells (2.2×10⁴ cells/ml) was added toa well of a 96-well microtiter plate containing 10 μl of a 10% DMSO inRPMI-1640 media solution containing 1 nM to 100 μM gambogic acid orother test compound (0.1 nM to 10 μM final). An aliquot of 90 μl ofcells was added to a well of a 96-well microtiter plate containing 10 μlof a 10% DMSO in RPMI-1640 media solution without compound as thecontrol sample for maximal cell proliferation (A_(max)). The sampleswere mixed by agitation and then incubated at 37° C. for 48 h in a 5%CO₂-95% humidity incubator. After incubation, the samples were removedfrom the incubator and 20 μl of CellTiter 96 AQ_(UEOUS) One SolutionCell Proliferation™ reagent (Promega) was added. The samples were mixedby agitation and incubated at 37° C. for 2-4 h in a 5% CO₂-95% humidityincubator. Using an absorbance plate reader (Model 1420 WallacInstruments), an initial reading (T=0) was made approximately 1-2 min.after addition of the solution, employing absorbance at 490 nm. Thisdetermines the possible background absorbance of the test compounds. Noabsorbance for gambogic acid or its analogs or derivatives was found at490 nm. After the 2-4 h incubation, the samples were read for absorbanceas above (Aest).

Baseline for GI₅₀ (dose for 50% inhibition of cell proliferation) andLC₅₀ (dose for 50% cell death) of initial cell numbers was determined byadding an aliquot of 90 μl of cells or 90 μl of media, respectively, towells of a 96-well microtiter plate containing 10 μl of a 10% DMSO inRPMI-1640 media solution. The samples were mixed by agitation and thenincubated at 37° C. for 0.5 h in a 5% CO₂-95% humidity incubator. Afterincubation, the samples were removed from the incubator and 20 μl ofCellTiter 96 AQ_(UEOUS) One Solution Cell Proliferation™ reagent(Promega) was added. The samples were mixed by agitation and incubatedat 37° C. for 2-4 h in a 5% CO₂-95% humidity incubator. Absorbance wasread as above, (A_(T=0)) defining absorbance for initial cell numberused as baseline in GI₅₀ determinations and (A_(min)) definingabsorbance for media alone used as baseline in LC₅₀ determinations.

Calculation:

GI₅₀ (dose for 50% inhibition of cell proliferation)

50=100×[(A_(Test)−A_(T=0))/(A_(max)−A_(T=0))]

LC₅₀ (dose for 50% cell death)

10=50 =100×[(A_(Test)−A_(min))/(A_(T=0)−A_(min))]

The GI₅₀ (nM) and LC₅₀ (nM) are summarized in Table III:

TABLE III GI₅₀ and LC₅₀ in Cancer Cells Methyl-6- Gambogic MethylGambogyl Methoxy- Gambogenic acid Gambogate Piperidine gambogate AcidGambogenin GI₅₀ GI₅₀ GI₅₀ GI₅₀ GI₅₀ GI₅₀ Cell lines (nM) LC₅₀ (nM) LC₅₀(nM) LC₅₀ (nM) LC₅₀ (nM) LC₅₀ (nM) LC₅₀ T-47D  65  450  40  50  50  50 50  80  500  500  500  500 ZR-75-1 400  500 300  500 300  500 400 500ND ND ND ND PC-3 500  700 500  500 500  500 500 500 3000 5000 5000 5000DU-145 500  800 500  500 500  500 500 900  600 5000 2000 5000 A-549 8005000 500 2000 800 5000 500 900 ND ND ND ND SHP-77 500  500 500  500 500 500 500 500 ND ND ND ND HL-60 500  700  50  500 100  800 100 800 ND NDND ND MRC-5 400  500 200  500 800  800 500 500  500 5000 3000 5000 ND =not determined

Thus, gambogic acid and its analogs and derivatives are identified aspotent antineoplastic compounds that both inhibit cell proliferation(GI50) and elicit cell death (LC₅₀).

EXAMPLE 92 Identification Of Vinblastine, Cisplatin, 5-Fluorouracil,Taxol, Camptothecin, Doxorubicin, Etoposide And Methotrexate AsConventional Antineoplastic Agents That Are Not Efficient CaspaseCascade Activators In Solid Tumors

T-47D, ZR-75-1, PC-3 and HL-60 cells were grown and harvested as inExample 90. An aliquot of 45 μl of cells was added to a well of a96-well microtiter plate containing 5 μl of a 10% DMSO in RPMI-1640media solution containing 100 μM of test compounds (10 μM final). Analiquot of 45 μl of cells was added to a well of a 96-well microtiterplate containing 5 μl of a 10% DMSO in RPMI-1640 media solution withouttest compound as the control sample. The samples were mixed by agitationand then incubated at 37° C. for 24 h in a 5% CO₂-95% humidityincubator. After incubation, the samples were removed from the incubatorand 50 μl of a solution containing 20 μM ofN-(Ac-DEVD)-N′-ethoxycarbonyl-R110 fluorogenic substrate (SEQ ID NO:1)(Cytovia, Inc.), 20% sucrose (Sigma), 20 mM DTT (Sigma), 200 mM NaCl(Sigma), 40 mM Na PIPES buffer pH 7.2 (Sigma), and 500 μg/mllysolecithin (Calbiochem) was added. The samples were mixed by agitationand incubated for 3 h at room temperature. Using a fluorescent platereader (Model 1420 Wallac Instruments), an initial reading (T=0) wasmade approximately 1-2 min. after addition of the substrate solution,employing excitation at 485 nm and emission at 530 nm, to determine thebackground fluorescence of the control sample. After the 3 h incubation,the samples were read for fluorescence as above (T=3 h).

Calculation:

The Relative Fluorescence Unit values (RFU) were used to calculate thesample readings as follows:

 RFU_((T=3hr))−Control RFU_((T=0))=Net RFU_((T=3hr))

The activity in caspase cascade activation was determined by the ratioof the net RFU value for test compounds to that of control samples. Aratio around 1 indicates that the compound is not an efficient caspasecascade activator. The ratios are summarized in Table IV.

TABLE IV Activity of Known Antineoplastic Compound as Caspase CascadeActivators Cell lines T-47D PC-3 Vinblastine 0.9 0.8 Cisplatin 1.1 0.95-fluorouracil 0.8 0.7 Taxol 0.9 0.7 Camptothecin 0.7 0.6 Doxorubicin1.3 1.1 Etoposide 1.0 0.8 Methotrexate 0.8 0.7

Thus, vinblastine, cisplatin, 5-fluorouracil, taxol, camptothecin,doxorubicin, etoposide and methotrexate are identified as knownantineoplastic compounds that are not caspase cascade activators in thisassay.

EXAMPLE 93

Morphological Change of T47D Cells Treated with Gambogic Acid Cellsundergoing apoptosis typically demonstrate several characteristicmorphological changes, including rounding and blebbing. In addition,apoptotic adherent cells in culture lose their ability to remainattached to the culture dish. The ability of gambogic acid to triggerthese morphological changes in T47D cells was investigated.

60 mm culture dishes were seeded with 750,000 T47D cells and thecultures were incubated under normal growth conditions (complete mediumwith 10% FBS) for 24 h. The cells were then treated with 2.5 μM ofgambogic acid and further incubated under normal growth conditions for 2or 6 h. Morphological changes were documented by photographing the cellsunder phase contrast illumination.

As shown in FIGS. 1A-C, T47D cells incubated with vehicle (Control) arephase-dark and show a normal, flat morphology (FIG. 1A). After 2 h oftreatment with gambogic acid, many of the cells have taken on a rounded,phase-bright morphology (FIG. 1B). By 6 h of treatment with gambogicacid, most of the cells in the culture are rounded up and are beginningto detach from the dish (FIG. 1C). At this timepoint, many of the cellsalso show evidence of blebbing. Based on these data, it was concludedthat gambogic acid induces apoptotic morphological changes in T47Dcells.

EXAMPLE 94 Gambogic Acid Induces Nuclear Fragmentation in T47D BreastCancer Cells

T47D cells were grown and plated as described in Example 90. The cellswere treated with 10 μM of gambogic acid and the plate was incubated forup to 24 h at 37° C. in a 5% CO₂-95% humidity incubator. At 24 h, thecells were incubated with a live cell nucleic acid stain, Syto16(Molecular Probes) which stains DNA. After 2 washes with PBS, cells wereexamined under a fluorescence microscope. The nuclear staining ofuntreated cells showed normal nuclei (FIG. 2A) whereas the gambogic acidtreated cells showed condensed and fragmented nuclei in a largepopulation of the cells (FIG. 2B). Nuclear fragmentation is a clearindicator of cellular apoptosis.

EXAMPLE 95 Gambogic Acid Induces Characteristic Apoptotic Morphology inJurkat Cells

Jurkat T leukemia cells were grown in RPMI 1640 media (LifeTechnologies, Inc.)+10% FCS (Sigma Chemical Company) in a 5% CO₂-95%humidity incubator at 37° C., and maintained at a cell density between 4and 8×10⁵ cells/ml. Cells were harvested at 200×g and resuspended at1-2×10⁶ cells/ml into RPMI 1640 media+10% FCS, and 3 ml of the cells wasdispensed in each of three wells of a 6-well plate. One of the wells wastreated with 10 μM caspase inhibitor cbz-Val-Asp-fmk (Cytovia, Inc.;WO99/18781) and the plate was incubated at 37° C. in a 5% CO₂-95%humidity incubator for 1 h prior to addition of gambogic acid. The wellswith and without the caspase inhibitor were treated with 10 μM gambogicacid. The third well was treated with solvent (control cells). The platewas incubated at 37° C. in a 5% CO₂-95% humidity incubator.

At 30 min. after addition of gambogic acid an aliquot of cells from eachwell was taken into the capillary slides and observed under aphase-contrast microscope.

The control cell samples showed normal cell morphology (FIG. 3A) whereasafter 30 min. treatment with gambogic acid the cells showed blebbing andcellular fragmentation (FIG. 3B), hallmarks of apoptosis. The presenceof caspase inhibitor prevented the morphological changes (FIG. 3C),indicating that the changes are due to activation of caspases in thecell.

EXAMPLE 96 Activation of Caspases by Gambogic Acid in T47D Breast CancerCell Line and in Normal Fibroblasts MRC-5

T47D cells and MRC-5 cells were maintained and harvested as described inExample 90. An aliquot of 45 μl of cells was added to each well of a96-well microtiter plate. To determine the dose response of gambogicacid for inducing caspase activity, 5 μl of 20 μM gambogic acid in RPMImedia was added to wells in triplicates. Two-fold serial dilutions weremade for the lower concentrations. After incubation for 2 h, the sampleswere removed from the 5% CO₂-95% humidity incubator and caspase activitywas determined by addition of a fluorogenic substrate as described inExample 90.

The dose response (FIG. 4) indicated that the human breast cancer cellline T47D is more sensitive to induction of caspase activity than anormal fibroblast cell line MRC-5 by gambogic acid. Therefore, there isa potential therapeutic index with gambogic acid treatment.

EXAMPLE 97 Gambogic Acid Induces Caspase Activity in a Variety of SolidTumor Cell Lines Which Is Inhibited by a Caspase Inhibitor

T47D, ZR-75, PC3, SHP-77 and A-549 cells were maintained and harvestedas described in Example 90. Cells were added to 96-well plates asdescribed in Example 90. The cells were treated with 10 μM gambogicacid, in the presence and in the absence of 10 μM caspase inhibitorcbz-Val-Asp-fink (Cytovia, Inc.; WO99/18781). The plate was incubated upto 24 h at 37° C. in a 5% CO₂-95% humidity incubator. Caspase activitywas determined by addition of a fluorogenic substrate as described inExample 90.

Gambogic acid induced caspase activity in a ratio of greater than 2.5(+) above untreated cell levels in all the tested cancer cell lines(Table IV). The caspase activity detected was inhibited by the caspaseinhibitor (+), confirming that the fluorescent signal was due to caspaseactivity.

TABLE IV Gambogic Acid as Caspase Inducers in Solid Tumor CellsInhibition of caspase activity Cell line Caspase activity by InhibitorT47D + + ZR-75 + + PC-3 + + SHP-77 + + A-549 + +

EXAMPLE 98 Induction of PARP Cleavage by Gambogic Acid in Human TumorCells

Cleavage of the enzyme poly(ADP)ribose polymerase (PARP) by caspase-3and related proteases is considered to be one of the molecular hallmarksof caspase-mediated apoptosis. Therefore, the ability of gambogic acidto induce PARP cleavage in four different human tumor cell lines (Jurkatcells, HL-60 cells, T47D cells and PC3 cells) was determined.

Cells were cultured in complete growth medium containing 10% FBS andtreated with gambogic acid at concentrations of 2.5 μM or 5 μM for 2 to4 h. Control cultures were treated with a drug vehicle (DMSO), or thewell-characterized apoptosis inducer, staurosporine. At the end of theapoptosis induction period, the cells were harvested, washed once withPBS, quick-frozen on dry ice, and stored at −80° C. The cells were thenlysed in a standard immunoblotting lysis buffer and samples of thelysates were electrophoresed on 4% to 20% gradient polyacrylamide gels.The proteins in the gels were then transferred to PVDF membranes andprobed with a commercially-available rabbit polyclonal antibody to PARP.

FIGS. 5A-D illustrate the results of these experiments. A 2 h treatmentwith 2.5 μM gambogic acid induced almost complete PARP cleavage in bothJurkat cells (FIG. 5A) and HL-60 cells (FIG. 5B). 2.5 μM gambogic acidwas as effective as 1 μM staurosporine, one of the most potent apoptosisinducers known. There was no PARP cleavage in cells treated with drugvehicle (DMSO) or another inactive control.

FIG. 5C shows the effect of gambogic acid on PARP cleavage in T47Dcells. Within 2 h of treatment, using 2.5 μM gambogic acid, moderateinduction of PARP cleavage is observed; almost complete PARP cleavage isobserved with 5 μM gambogic acid. Within 4 h of treatment, bothconcentrations of gambogic acid give almost complete PARP cleavage.Under the same conditions, no cleavage of PARP was observed for cellstreated with 1 μM staurosporine.

PC3 cells, a human prostate cancer cell line, were more resistant to theinduction of PARP cleavage by gambogic acid (FIG. 5D). Within 2 h oftreatment, neither concentration (2.5 μM and 5 μM) of drug waseffective. Within 4 h of treatment, a moderate amount of PARP cleavageproduct could be observed with the highest dose of gambogic acid (5 μM).Under the same conditions, no cleavage of PARP was observed for cellstreated with 1 μM staurosporine.

Based on these experiments, it was concluded that gambogic acid triggersPARP cleavage in all four human tumor cell lines tested. These resultsindicate that gambogic acid is an effective inducer of caspase-mediatedapoptosis in tumor cells under normal growth conditions.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1 1 4 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 1 Asp Glu Val Asp 1

What Is claimed is:
 1. A method of inducing cell apoptosis in a mammalin need thereof, comprising administering to said mammal an effectiveamount of a compound having one of the Formulae I-III:

or a pharmacuetically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is optionally substituted lower alkyl;R_(b) and R_(c) are independently hydrogen, optionally substitutedheteroalkyl, optionally substituted lower alkyl, optionally substitutedaryl, optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(a) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b), and R_(a) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is hydrogen orprenyl; R₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino; and R₅ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b), R_(c) and R_(d) are defined above; withthe provisos that when said mammal has cancer, then (a) said compound isnot gambogic acid, isogambogic acid, morellinol, isomorellinol orgambogin, (b) said compound is of Formula I or II, and (c) R₃ is prenyl.2. The method of claim 1, wherein the dotted lines between C-9 and C-10of a compound of Formula I or III represent a double bond, R₄ is not acycloalkyl group, the other dotted lines are not epoxy groups, and R_(b)and R^(c) are not heteroalkyl groups.
 3. A method according to claim 1,wherein R₁ is formyl, acetyl, propionyl, carboxy, methoxycarbonyl,ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2(dimethylamino)-ethylcarbamyl or N-morpholinylcarbonyl, and the dottedlines represent double bonds.
 4. A method according to claim 1, whereinR₂ is hydrogen, formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethyl-carbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 5. A method according toclaim 1, wherein R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy,hydrogen, methoxy, ethoxy, methylthio, ethylthio, butylthio,dimethylamino, diethylamino, piperidinyl, pyrrolidinyl, imidazolyl,pyrazolyl, N-methylpiperazinyl, 2-(dimethylamino)ethylamino ormorpholinyl, and the dotted lines represent double bonds.
 6. A methodaccording to claim 1, wherein R₅ is hydrogen, acetyl, dimethylcarbamyl,diethylcarbamyl, 2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinyl-carbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl. 7.The method according to claim 1, wherein said compound is selected fromthe group consisting of: Methyl gambogate ester; 9,10-Dihydrogambogyl(4-methylpiperazine); 9,10-Dihydrogambogyl (2-dimethylaminoethylamine);9,10-Dihydro-12-hydroxygambogic acid; Gambogyl diethylamine; Gambogyldimethylamine; Gambogyl amine; Gambogyl hydroxyamine; Gambogylpiperidine; 6-Methoxy-gambogic acid; 6-(2-Dimethylaminoethoxy)-gambogicacid; 6-(2-Piperidinylethoxy)-gambogic acid;6-(2-Morpholinylethoxy)-gambogic acid; 6-Methoxy-gambogyl piperidine;Gambogyl morpholine; Gambogyl (2-dimethylaminoethylamine);9,10-Dihydro-10-morpholinyl-gambogyl morpholine;9,10-Dihydro-10-morpholinyl-gambogyl piperidine;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl piperidine;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl morpholine;9,10-Dihydro-10-piperidinyl-gambogyl piperidine;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl (4-methylpiperazine);10-Cyclohexyl-9,10-dihydrogambogic acid; 9,10-Dihydro-10-methyl gambogicacid; Gambogyl (4-methylpiperazine); Methyl-6-Methoxy-gambogate;9,10-Dihydro-10-methoxy-gambogic acid; 10-Butylthio-9,10-dihydrogambogicacid; 9,10-Dihydro-10-(4-methylpiperazinyl)-gambogic acid;9,10-Dihydro-10-pyrrolidinyl-gambogic acid;Methyl-9,10-dihyrdro-10-morpholinyl-gambogate;9,10-Dihydro-10-piperidinyl-gambogic acid;9,10-Dihydro-10-morpholinyl-gambogic acid;N-(2-Gambogylamido-ethyl)biotinamide; Gambogyl(2-(4-morpholinyl)ethylamine); 9,10-Epoxygambogic acid; Gambogyl(4-(2-pyridyl)piperazine);9,10-Dihydro-10-(4-(2-pyridyl)piperazinyl)gambogyl(4-(2-pyridyl)piperazine); 6-Acetylgambogic acid;9,10-Dihydro-10-(4-(2-pyridyl)piperazinyl)gambogic acid;8-(Gambogylamido)octanoic acid; 6-(Gambogylamido)hexanoic acid;12-(Gambogylamido)dode canoic acid ; 9,10-Dihydro-10-methoxy-gambogylpiperidine; Gambogyl (4-(2-pyrimidyl)piperazine); Gambogyl(bis(2-pyridylmethyl)amine); Gambogyl(N-(3-pyridyl)-N-(2-hydroxybenzyl)amine); Gambogyl (4-benzylpiperazine);Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine); Gambogyl(-methyl-5-(methylamino)-3-oxapentylamine); Gambogyl(N-methyl-8-(methylamino)-3,6-dioxaoctylamine); Gambogyl(N-ethyl-2-(ethylamino)ethylamine); Gambogyl (4-isopropylpiperazine);Gambogyl (4-cyclopentylpiperazine); Gambogyl(N-(2-oxo-2-ethoxyethyl)-(2-pyidyl)methylamine); Gambogyl(2,5-dimethylpiperazine); Gambogyl (3,5-dimethylpiperazine); Gambogyl(4-(4-acetylphenyl)piperazine); Gambogyl (4-ethoxycarbonylpiperazine);Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine); Gambogyl(4-(2-hydroxyethyl)piperazine); Gambogyl(N-methyl-2-(methylamino)ethylamine); Gambogyl(N-methyl-2-(benzylamino)ethylamine); Gambogyl(N-methyl-(6-methyl-2-pyridyl)methylamine); Gambogyl(N-ethyl-2-(2-pyridyl)ethylamine); Gambogyl(N-methyl-(2-pyridyl)methylamine); Gambogyl(N-methyl-4-(3-pyridyl)butylamine); Gambogyl(bis(3-pyridylmethyl)amine); Gambogyl (2,4-dimethyl-2-imidazoline);Gambogyl (4-methyl-homopiperazine); Gambogyl(4-(5-hydroxy-3-oxapentyl)piperazine); Gambogyl(3-dimethylaminopyrrolidine); Gambogyl ((2-furanyl)methylamine);Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine); Gambogyl(3,4,5-trimethoxybenzylamine); Gambogyl (2-(²-methoxyphenyl)ethylamine);Gambogyl (2-methoxybenzylamine); Gambogyl(3,4-methylenedioxybenzylamine); Gambogyl(2-(2,5-dimethoxyphenyl)ethylamine); Gambogyl(2-(3-methoxyphenyl)ethylamine); Gambogyl (3-(piperidinyl)propylamine);Gambogyl (2-(piperidinyl)ethylamine); Gambogyl(3,4-dimethoxybenzylamine); Gambogyl ((2-tetrahydrofuranyl)methylamine);Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine); Gambogyl(2-diethylaminoethylamine); Gambogyl(2,2-dimethyl-3-dimethylaminopropylamine); Gambogyl((N-ethoxycarbonyl-4-piperidinyl)amine); Gambogyl(2-carbamylpyrrolidine); Gambogyl (3-(homopiperidinyl)propylamine);Gambogyl ((N-benzyl-4-piperidinyl)amine); Gambogyl(2-(4-methoxyphenyl)ethylamine); Gambogyl (4-oxa-hex-5-enylamine);Ganbogyl (6-hydroxyhexylamine); Gambogyl(2-(3,5-dimethoxyphenyl)ethylamine); Gambogyl(3,5-dimethoxybenzylamine); and Gambogyl(2-carbamyl-2-(4-hydroxyphenyl)ethylamine).
 8. A method for treatingcancer, comprising administering to an animal in need of such treatmentan effective amount of a compound having the Formula I or II:

or a pharmacuetically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds or epoxy groups; X together with theattache carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is optionally substituted lower alkyl;R_(b) and R_(a) are independently hydrogen, optionally substitutedheteroalkyl, optionally substituted lower alkyl, optionally substitutedaryl, optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(a)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b) and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is prenyl; andR₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino; with the proviso that saidcompound is not gambogic acid, isogambogic acid, morellinol,isomorellinol or gambogin.
 9. The method of claim 8, wherein in thecompound the dotted lines between C-9 and C-10 of a compound of FormulaI or III represent a double bond, R₄ is not a cycloalkyl group, theother dotted lines are not epoxy groups, and R_(b) and R_(c) are notheteroalkyl groups.
 10. The method according to claim 8, wherein themethod is for treating Hodgkin's disease, non-Hodgkin's lymphomas, acuteand chronic lymphocytic leukemias, multiple myeloma, neuroblastoma,breast carcinomas, ovarian carcinomas, lung carcinomas, Wilms'tumor,cervical carcinomas, testicular carcinomas, soft-tissue sarcomas,chronic lymphocytic leukemia, primary macroglobulinemia, bladdercarcinomas, chronic granulocytic leukemia, primary brain carcinomas,malignant melanoma, small-cell lung carcinomas, stomach carcinomas,colon carcinomas, malignant pancreatic insulinoma, malignant carcinoidcarcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides,head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas,acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroidcarcinomas, esophageal carcinomas, malignant hypercalcemia, cervicalhyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemiavera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer,or prostatic carcinomas.
 11. A method for the treatment of drugresistant cancer, comprising administering to an animal in need of suchtreatment an effective amount of a compound having one of the FormulaeI-III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is optionally substituted lower alkyl;R_(b) and R_(c) are independently hydrogen, optionally substitutedheteroalkyl, optionally substituted lower alkyl, optionally substitutedaryl, optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b) and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is hydrogen orprenyl; R₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino; and R₅ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b), R_(c) and R_(d) are defined above; withthe proviso that said compound is not gambogic acid, isogambogic acid,morellinol, or isomorellinol.
 12. The method of claim 11, wherein in thecompound the dotted lines between C-9 and C-10 of a compound of FormulaI or III represent a double bond, R₄ is not a cycloalkyl group, theother dotted lines are not epoxy groups, and R_(b) and R_(c) are notheteroalkyl groups.
 13. The method according to claim 8 or 11, whereinsaid compound is administered together with at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent.
 14. The method according to claim 13, wherein said known cancerchemotherapeutic agent is selected from the group consisting ofbusulfan, cis-platin, mitomycin C, carboplatin, colchicine, vinblastine,paclitaxel, docetaxel, camptothecin, topotecan, doxorubicin, etoposide,5-azacytidine, 5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine,ara-C, hydroxyurea, thioguanine, melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, Herceptin® (trastuzumab), Rituxan®(rituximab) and alanosine.
 15. The method according to claim 8 or 11,wherein said animal is also treated with radiation-therapy.
 16. Themethod according to claim 8 or 11, wherein said compound(s) areadministered after surgical treatment for cancer.
 17. The methodaccording to claim 1, wherein the mammal has an autoimmune disease. 18.The method according to claim 1, wherein the mammal has rheumatoidarthritis.
 19. The method according to claim 1, wherein the mammal hasinflammation or inflammatory bowel disease.
 20. The method according toclaim 1, wherein the mammal has a skin disease.
 21. The method accordingto claim 20, wherein said skin disease is psoriasis.
 22. A compoundhaving the Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is optionally substituted lower alkyl;R_(b) and R_(c) are independently hydrogen, optionally substitutedheteroalkyl, optionally substituted lower alkyl, optionally substitutedaryl, optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b) and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; and R₃ is prenyl;with the proviso that if R₁ is methyl, carboxy or methoxycarbonyl, and Xand Y are O, then R₂ is not a hydrogen, acetyl or methyl.
 23. A compoundof claim 22, wherein the dotted lines between C-9 and C-10 represent adouble bond, the other dotted lines are not epoxy groups, and R_(b) andR_(a) are not heteroalkyl groups.
 24. A compound according to claim 22,wherein R₁ is formyl, acetyl, propionyl, carboxy, methoxycarbonyl,ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)-ethylcarbamyl or N-morpholinylcarbonyl, and the dottedlines represent double bonds.
 25. A compound according to claim 22,wherein R₂ is hydrogen, formyl, acetyl, dimethylcarbamyl,diethylcarbamyl, 2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 26. A compound having theFormula II:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is optionally substituted lower alkyl;R_(b) and R_(c) are independently hydrogen, optionally substitutedheteroalkyl, optionally substituted lower alkyl, optionally substitutedaryl, optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b) and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is hydrogen orprenyl; and R₄ is hydrogen, halogen, hydroxy, optionally substitutedalkyl, cycloalkyl, alkoxy, alkylthio or amino; with the proviso that ifR₁ is formyl or carboxy, R₂ is hydrogen, R₃ is hydrogen, and X and Y areO, then R₄ is not a hydrogen, hydroxy, methoxy or ethoxy.
 27. A compoundof claim 26, wherein R₄ is not a cycloalkyl group, the dotted lines arenot epoxy groups, and R_(b) and R_(c) are not heteroalkyl groups.
 28. Acompound according to claim 26, wherein R₁ is formyl, acetyl, propionyl,carboxy, methoxycarbonyl, ethoxycarbonyl, methylthiocarbonyl,ethylthiocarbonyl, butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)-ethylcarbamyl or N-morpholinylcarbonyl, and the dottedlines represent double bonds.
 29. A compound according to claim 26,wherein R₂ is hydrogen, formyl, acetyl, dimethylcarbamyl,diethylcarbamyl, 2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 30. A compound according toclaim 26, wherein R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy,hydrogen, methoxy, ethoxy, methylthio, ethylthio, butylthio,dimethylamino, diethylamino, piperidinyl, pyrrolidinyl, imidazolyl,pyrazolyl, N-methylpiperazinyl, 2-(dimethylamino)ethylamino ormorpholinyl, and the dotted lines represent double bonds.
 31. A compoundaccording to claim 22, wherein said compound is selected from the groupconsisting of: Gambogyl dimethylamine; Gambogyl amine; Gambogyldiethylamine; Gambogyl hydroxyamine; Gambogyl piperidine;6-Methoxy-gambogic acid; 6-(2-Dimethylaminoethoxy)-gambogic acid;6-(2-Piperidinylethoxy)-gambogic acid; 6-(2-Morpholinylethoxy)-gambogicacid; 6-Methoxy-gambogyl piperidine; Gambogyl morpholine; Gambogyl(2-dimethylaminoethylamine); Gambogyl (4-methylpiperazine);N-(2-Gambogylamido-ethyl)biotinamide; Gambogyl(2-(4-morpholinyl)ethylamine); Gambogyl (4-(2-pyridyl)piperazine);6-Acetylgambogic acid; N-Hydroxysuccinimidyl gambogate;8-(Gambogylamido)octanoic acid; 6-(Gambogylamido)hexanoic acid;12-(Gambogylamido)dodecanoic acid;N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate; Gambogyl(4-(2-pyrimidyl)piperazine); Gambogyl (bis(2-pyridylmethyl)amine);Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine); Gambogyl(4-benzylpiperazine); Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine); Gambogyl(N-methyl-8-(methylamino)-3,6-dioxaoctylarnine); Gambogyl(N-ethyl-2-(ethylamino)ethylamine); Gambogyl (4-isopropylpiperazine);Gambogyl (4-cyclopentylpiperazine); Gambogyl(N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine); Gambogyl(2,5-dimethylpiperazine); Gambogyl (3,5-dimethylpiperazine); Gambogyl(4-(4-acetylphenyl)piperazine); Gambogyl (4-ethoxycarbonylpiperazine);Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine); Gambogyl(4-(2-hydroxyethyl)piperazine); Gambogyl(N-methyl-2-(methylamino)ethylamine); Gambogyl(N-methyl-2-(benzylamino)ethylamine); Gambogyl(N-methyl-(6-methyl-2-pyridyl)methylamine); Gambogyl(N-methy-2-(2-pyridyl)ethylamine); Gambogyl(N-methyl-(2-pyridyl)methylamine); Gambogyl(N-methyl-4-(3-pyridyl)butylamine); Gambogyl(bis(3-pyridylmethyl)amine); Gambogyl (2,4-dimethyl-2-imidazoline);Gambogyl (4-methyl-homopiperazine); Gambogyl(4-(5-hydroxy-3-oxapentyl)piperazine); Gambogyl(3-dimethylaminopyrrolidine); Gambogyl ((2-furanyl)methylamine);Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine); Gambogyl(3,4,5-trimethoxybenzylamine); Gambogyl (2-(2-methoxyphenyl)ethylamine);Gambogyl (2-methoxybenzylamine); Gambogyl(3,4-methylenedioxybenzylamine); Gambogyl(2-(2,5-dimethoxyphenyl)ethylamine); Gambogyl(2-(3-methoxyphenyl)ethylamine); Gambogyl (3-(piperidinyl)propylamine);Gambogyl (2-(piperidinyl)ethylamine); Gambogyl(3,4-dimethoxybenzylamine); Gambogyl ((2-tetrahydrofuranyl)methylamine);Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine); Gambogyl(2-diethylaminoethylamine); Gambogyl(2,2-dimethyl-3-dimethylaminopropylamine); Gambogyl((N-ethoxycarbonyl-4-piperidinyl)amine); Gambogyl(2-carbamylpyrrolidine); Gambogyl (3-(homopiperidinyl)propylamine);Gambogyl ((N-benzyl-4-piperidinyl)amine); Gambogyl(2-(4-methoxyphenyl)ethylamine); Gambogyl (4-oxa-hex-5-enylamine);Ganbogyl (6-hydroxyhexylamine); Gambogyl(2-(3,5-dimethoxyphenyl)ethylamine); Gambogyl(3,5-dimethoxybenzylamine); and Gambogyl(2-carbamyl-2-(4-hydroxyphenyl)ethylamine).
 32. A compound according toclaim 26, wherein said compound is selected from the group consistingof: 9,10-Dihydrogambogyl (4-methylpiperazine); 9,10-Dihydrogambogyl(dimethylamino)ethylamine; 9,10-Dihydro-1 2-hydroxygambogic acid;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl piperidine;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl morpholine;9,10-Dihydro-10-piperidinyl-gambogyl piperidine;9,10-Dihydro-10-(4-methylpiperazinyl)-gambogyl (4-methylpiperazine);9,10-Dihydro-10-(4-methylpiperazinyl)-gambogic acid;9,10-Dihydro-10-pyrrolidinyl-gambogic acid;Methyl-9,10-dihydro-10-morpholinyl-gambogate;9,10-Dihydro-10-morpholinyl-gambogyl morpholine;9,10-Dihydro-10-morpholinyl-gambogyl piperidine;9,10-Dihydro-10-methoxy-gambogic acid; 10-Butylthio-9,10-dihydrogambogicacid; 9,10-Dihydro-10-piperidinyl-gambogic acid;9,10-Dihydro-10-morpholinyl-gambogic acid;10-Cyclohexyl-9,10-dihydro-gambogic acid;9,10-Dihydro-10-methyl-gambogic acid; 9,10-Dihydro-10-methoxy-gambogylpiperidine; 9,10-Dihydro-10-(4-(2-pyridyl)piperazinyl)gambogyl(4-(2-pyridyl)piperazine);9,10-Dihydro-10-(4-(2-pyridyl)piperazinyl)gambogic acid; and9,10-Epoxygambogic acid.
 33. A pharmaceutical composition, comprising acompound of claim 22 or 26, and a pharmaceutically acceptable carrier.34. The pharmaceutical composition of claim 33, further comprising atleast one known cancer chemotherapeutic agent, or a pharmaceuticallyacceptable salt of said agent.
 35. The pharmaceutical composition ofclaim 34, wherein said known cancer chemotherapeutic agent is selectedfrom the group consisting of busulfan, cis-platin, mitomycin C,carboplatin, colchicine, vinblastine, paclitaxel, docetaxel,caiptothecin, topotecan, doxorubicin, etoposide, 5-azacytidine,5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine, ara-C,hydroxyurea, thioguanine, meiphalan, chorambucil, cyclophosamide,ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin,bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoicacid, tamoxifen, Herceptin® (trastuzumab), Rituxan® (rituximab) andalanosine.
 36. The method of claim 1, wherein said compound is ofFormula I.
 37. The method of claim 36, wherein R₃ is prenyl.
 38. Themethod of claim 1, wherein said compound is of Formula II.
 39. Themethod of claim 38, wherein R₃ is prenyl.
 40. The method of claim 1,wherein said compound is of Formula III.
 41. The method of claim 40,wherein R₃ is prenyl.
 42. The method of claim 1, with the furtherproviso that said disorder is other than cancer, (a) said compound isnot gambogic acid, isogambogic acid, morellinol, isomorellinol orgambogin, (b) the compound is of Formula I or II, and (c) R₃ is prenyl.43. The method of claim 1, wherein said disorder is other than cancer.44. The method of claim 8, wherein said compound is of Formula I. 45.The method of claim 8, wherein said compound is of Formula II.
 46. Themethod of claim 11, wherein said compound is of Formula I.
 47. Themethod of claim 11, wherein said compound is of Formula II.
 48. Themethod of claim 11, wherein (a) said compound is of Formula I or II, and(b) R₃ is prenyl.
 49. The compound of claim 26, wherein R₃ is prenyl.50. The compound of claim 26, wherein Y is O.
 51. A method of inducingcell apoptosis in a mammal in need thereof, comprising administering tosaid mammal an effective amount of a compound having the Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is optionallysubstituted alkylthiocarbonyl, optionally substituted aminocarbonyl(carbamyl, R_(b)R_(c)NCO) or hydroxyaminocarbonyl, where R_(b) and R_(c)are independently hydrogen, optionally substituted heteroalkyl,optionally substituted aryl, optionally substituted heteroaryl oroptionally substituted lower aralkyl groups; or R_(b) and R_(c) may betaken together with the attached N to form an optionally substituted,saturated or partially saturated 5-7 membered heterocyclo group; andwhen at least one of X and Y together with the attached carbon is amethylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; then R₁ is additionallyoptionally substituted lower alkyl, formyl, methylenehydroxy, carboxy,acyl (R_(a)CO), or optionally substituted alkoxycarbonyl (R_(a)OCO),where R_(a) is optionally substituted lower alkyl; R₂ is hydrogen,optionally substituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO)or sulfonyl (R_(d)SO₂), where R_(a), R_(b), and R_(c) are defined above;R_(d) is hydrogen, optionally substituted lower alkyl, optionallysubstituted aryl, or optionally substituted lower aralkyl groups; and R₃is hydrogen or prenyl.
 52. A method of inducing cell apoptosis in amammal in need thereof, comprising administering to said mammal aneffective amount of a compound having the Formula II:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is optionallysubstituted alkylthiocarbonyl, optionally substituted aminocarbonyl(carbamyl, R_(b)R_(c)NCO) or hydroxyaminocarbonyl, where R_(b) and R_(c)are independently hydrogen, optionally substituted heteroalkyl,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b), and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is hydrogen orprenyl; and R₄ is hydrogen, halogen, hydroxy, optionally substitutedalkyl, cycloalkyl, alkoxy, alkylthio or amino.
 53. A method of inducingcell apoptosis in a mammal in need thereof comprising administering tosaid mammal an effective amount of a compound having the Formula II:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is optionallysubstituted alkylthiocarbonyl, optionally substituted aminocarbonyl(carbamyl, R_(b)R_(c)NCO) or hydroxyaminocarbonyl, where R_(b) and R_(c)are independently hydrogen, optionally substituted heteroalkyl,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group; R₂ is hydrogen, optionallysubstituted alkyl, acyl (R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl(R_(d)SO₂), where R_(a), R_(b), and R_(c) are defined above; R_(d) ishydrogen, optionally substituted lower alkyl, optionally substitutedaryl, or optionally substituted lower aralkyl groups; R₃ is hydrogen orprenyl; R₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino; and when R₄ is halogen,cycloalkyl, alkylthio or amino, then R₁ additionally is optionallysubstituted lower alkyl, formyl, methylenehydroxy, carboxy, acyl(R_(a)CO), or optionally substituted alkoxycarbonyl (R_(a)OCO), whereR_(a) is optionally substituted lower alkyl.
 54. The method of any oneof claims 51-53, wherein said disorder is cancer.